Activatable fibrinolytic and anti-thrombotic proteins

ABSTRACT

Proteinaceous compounds are activatable by enzymes of the clotting cascade to have fibrinolytic or clot formation inhibition activity. For example, a plasminogen analogue is activatable to plasmin by thrombin or Factor Xa. Fibrinolytic or clot formation inhibition activity is therefore directed to the site of clot formation.

This invention relates to proteinaceous compounds which can be activatedto have fibrinolytic activity or to inhibit blood clot formation. Italso relates to nucleic acid (DNA and RNA) coding for all or part ofsuch compounds. In preferred embodiments, the invention relates toplasminogen analogues, their preparation, pharmaceutical compositionscontaining them and their use in the treatment of thrombotic disease.

Plasminogen is a key component of the fibrinolytic system which is thenatural counterpart to the clotting system in the blood. In the processof blood coagulation, a cascade of enzyme activities are involved ingenerating a fibrin network which forms the framework of a clot, orthrombus. Degradation of the fibrin network (fibrinolysis) isaccomplished by the action of the enzyme plasmin. Plasminogen is theinactive precursor of plasmin and conversion of plasminogen to plasminis accomplished by cleavage of the peptide bond between arginine 561 andvaline 562 of plasminogen. Under physiological conditions this cleavageis catalysed by tissue-type plasminogen activator (tPA) or byurokinase-type plasminogen activator (uPA).

If the balance between the clotting and fibrinolytic systems becomeslocally disturbed, intravascular clots may form at inappropriatelocations leading to conditions such as coronary thrombosis andmyocardial infarction, deep vein thrombosis, stroke, peripheral arterialocclusion and embolism. In such cases, the administration offibrinolytic agents has been shown to be a beneficial therapy for thepromotion of clot dissolution.

Fibrinolytic therapy has become relatively widespread with theavailability of a number of plasminogen activators such as tPA, uPA,streptokinase and the anisoylated plasminogen streptokinase activatorcomplex, APSAC. Each of these agents has been shown to promote clotlysis, but all have deficiencies in their activity profile which makesthem less than ideal as therapeutic agents for the treatment ofthrombosis (reviewed by Marder and Sherry, New England Journal ofMedicine 1989, 318: 1513-1520). One of the major problems with tPA forthe treatment of acute myocardial infarction or other thromboticdisorders is that it is rapidly cleared from the circulation with aplasma half-life in man of around 5 minutes (Bounameaux et al in:"Contemporary Issues in Haemostasis and Thrombosis" vol 1 p5-91, 1985.Collen et al eds, Churchill Livingstone). This results in the need toadminister tPA by infusion in large doses. The treatment is thereforeexpensive and is delayed as the patient has to be hospitalised beforetreatment can commence. Urokinase, in either the single chain form(scuPA) or the two chain form (tcuPA), has a similar rapid plasmaclearance and also requires administration by continuous infusion.

A major problem shared by all of these agents is that at clinicallyuseful doses, they are not thrombus specific as they activateplasminogen in the general circulation. The principal consequence ofthis is that proteins such as fibrinogen involved in blood clotting aredestroyed and dangerous bleeding can occur. This also occurs with tPAdespite the fact that, at physiological concentrations, it binds tofibrin and shows fibrin selective plasminogen activation.

Another important shortcoming in the performance of existing plasminogenactivators is that re-occlusion of the reperfused blood vessel commonlyoccurs after cessation of administration of the thrombolytic agent. Thisis thought to be due to the persistence of thrombogenic material at thesite of thrombus dissolution.

An alternative approach to enhancing fibrinolysis has now been devisedwhich is based on the use of molecules activatable to have fibrinolyticactivity or to inhibt clot formation. The activation (which may involvecleavage) can be catalysed by one or more endogenous enzymes involved inblood clotting. An advantage of this approach is that thrombusselectivity of fibrinolytic or inhibition of clot formation activity isachieved by way of the thrombus-specific localisation of the activatingenzymes.

According to a first aspect of the present invention, there is provideda proteinaceous compound which is activatable, by an enzyme involved inblood clotting, to have fibrinolytic activity or to inhibit clotformation.

Proteinaceous compounds in accordance with the first aspect of theinvention, are therefore activatable in at least one of two ways. First,a compound may be activated to have fibrinolytic activity. Secondly, acompound may be activated to inhibit clot formation. Conceivably, acompound may be activatable to have both functions. Activation is mostconveniently achieved by cleavage, in many cases.

Preferably the compound, when activated, has substantially the samequalitative activity as a natural mammalian fibrinolytic agent and/or amammalian inhibitor of clot formation. In quantitative terms, while itis preferred that the activity be as good as, if not better than, thenatural compound, the benefits of the invention may still be had if theactivity is not as good. It will be understood that preferred compoundsof the invention may therefore have the same qualitative activity as anatural precursor of a natural mammalian fibrinolytic agent and/or amammalian inhibitor of clot formation. Again, in quantitative terms, thefacility with which the precursor can be activated is preferably, butneed not necessarily be, as good as the natural compound.

A natural proteinaceous compound which is activatable to havefibrinolytic activity is plasminogen, which is cleaved to form plasmin.Plasminogen analogues form a preferred group of compounds of thisinvention.

Analysis of the wild-type plasminogen molecule has revealed that it is aglycoprotein composed of a serine protease domain, five kringle domainsand an N-terminal sequence of 78 amino acids which may be removed byplasmin cleavage. Cleavage by plasmin involves hydrolysis of theArg(68)-Met(69), Lys(77)-Lys(78) or Lys(78)-Val(79) bonds to createforms of plasminogen with an N-terminal methionine, lysine or valineresidue, all of which are commonly designated as lys-plasminogen. Intactplasminogen is referred to as glu-plasminogen because it has anN-terminal glutamic acid residue. Glycosylation occurs on residuesAsn(289) and Thr(346) but the extent and composition are variable,leading to the presence of a number of different molecular weight formsof plasminogen in the plasma. The serine protease domain can berecognised by its homology with other serine proteases and on activationto plasmin is the catalytically active domain involved in fibrindegradation. The five kringle domains are homologous to those in otherplasma proteins such as tPA and prothrombin and are involved in fibrinbinding and thus localisation of plasminogen and plasmin to thrombi.Plasminogen is a zymogen which normally circulates in the blood as asingle polypeptide chain and is converted to the two-chain enzymeplasmin by cleavage of a peptide bond between amino acids 561 (arg) and562 (val). This cleavage is catalysed specifically by plasminogenactivators such as tPA and uPA. This is reviewed in: Castellino, F. J.,1984, Seminars in Thrombosis and Haemostasis 10; 18-23. In thisspecification, plasminogen is numbered according to the proteinsequencing studies of Sottrup-Jensen et al (in: Atlas of ProteinSequence and Structure (Dayhoff, M. O., ed.) 5 suppl. 3, p.95 (1978))which indicated that plasminogen was a 790 amino acid protein and thatthe site of cleavage was the Arg(560)-Val(561) peptide bond. However, asuitable plasminogen cDNA useful in this embodiment of the invention andthat isolated by Forsgren et al (FEBS Letters 213 254-260 (1987)) codefor a 791 residue protein with an extra Ile at position 65. In thisspecification, the numbering of the amino acids in plasminogencorresponds to that of the cDNA used. There may be polymorphism in thestructure of plasminogen and there may be forms of plasminogen in whichthe numbering of the cleavage site differs but it is intended that suchvariants be included in the embodiment.

Therefore the term "plasminogen analogue", as used in thisspecification, means a molecule differing from wild type plasminogen andhaving the ability to be cleaved or otherwise acted on to form amolecule having plasmin activity.

The plasma half-life of glu-plasminogen has been determined to be 2.2days and that of lys-plasminogen to be 0.8 days (Claeys, H. andVermylen, J. 1974. Biochim. Biophys. Acta 342: 351-359; Wallen, P. andWiman, B. in: "Proteases and Biological Control", 291-303. Reich, E. etal. eds, Cold Spring Harbor Laboratory).

Plasminogen analogues within the scope of this embodiment of theinvention retain the fibrin binding activity of wild type plasminogen toan adequate degree but have altered activation characteristics;preferred plasminogen analogues have a plasma half life which is atleast that of wild type plasminogen, but this property is not essential.

The blood coagulation mechanism comprises a series of enzyme reactionswhich culminate in the production of insoluble fibrin, which forms themesh-like protein framework of blood clots. Thrombin is the enzymeresponsible for the conversion of soluble fibrinogen to fibrin.Conversion of prothrombin, the inactive precursor of thrombin, tothrombin is catalysed by activated Factor X (Factor Xa). (Thrombin isalso known as Factor IIa, and prothrombin as Factor II.)

Factor Xa is generated from Factor X extrinsically or intrinsically. Inthe extrinsic route, Factor VII is activated to Factor VIIa, whichgenerates Factor Xa from Factor X. In the intrinsic route, theactivation of Factor X to Factor Xa is catalysed by Factor IXa. FactorIXa is generated from Factor IX by the action of Factor XIa, which inturn is generated by the action of Factor XIIa on Factor XI. Factor XIIais generated from Factor XII by the action of Kallikrein. Factors VIIIaand Va are thought to act as cofactors in the activation of Factors Xand II, respectively.

Fibrin, as first formed from fibrinogen, is in the loose form. Loosefibrin is converted to tight fibrin by the action of Factor XIIIa, whichcrosslinks fibrin molecules.

Activated protein C is an anticoagulant serine protease generated in thearea of clot formation by the action of thrombin, in combination withthrombomodulin, on protein C. Activated protein C regulates clotformation by cleaving and inactivating the pro-coagulant cofactors Vaand VIIIa.

The term "enzyme involved in blood clotting" as used in thisspecification therefore includes kallikrein Factors XIIa, XIa, IXa,VIIa, Xa and thrombin (Factor IIa), which are directly involved in theformation of fibrin and activated protein C, which is involved in thecontrol of blood clotting. The most preferred enzymes are Factor Xa andthrombin because they are most immediately involved with fibrinformation.

Generation and activity of at least Factor Xa and thrombin is tightlyregulated to ensure that thrombus generation is restricted to the siteof the thrombogenic stimulus. This localisation is achieved by thecombined operation of at least two control mechanisms: the bloodclotting enzymes function as complexes intimately associated with thephospholipid cellular membranes of platelets and endothelial cells atthe site of vascular injury (Mann, K. G., 1984, in: "Progress inHemostasis and Thrombosis", 1-24, ed Spaet, T. H. Grune and Stratton);and, free thrombin or Factor Xa released from the thrombus site into thecirculation is rapidly inactivated by the action of proteinaseinhibitors such as antithrombin III.

Thus, the activity of the penultimate (Factor Xa) and the final(thrombin) enzymes in the clotting cascade are particularly welllocalised to the site of thrombus generation and for this reason arepreferred.

Thrombin has been found to remain associated with thrombi and to bindnon-covalently to fibrin. On digestion of thrombi with plasmin, activethrombin is liberated and is thought to contribute to the reformation ofthrombi and the re-occlusion of vessels which commonly occurs followingthrombolytic treatment with plasminogen activators (Bloom A. L., 1962,Br. J. Haematol, 82, 129; Francis et al, 1983, J. Lab. Clin. Med., 102,220; Mirshahi et al, 1989, Blood 74, 1025).

For these reasons, it is preferred in certain embodiments of theinvention to modify plasminogen or another potentially activatableproteinaceous compound to make it activatable by thrombin or Factor Xathereby to create a preferred class of thrombus-selective, fibrinolyticor clot formation inhibiting proteins. The most preferred plasminogenanalogues retain the favourable property of the parent plasminogenmolecule of possessing a long plasma half-life and exhibit thrombusselectivity by a combination of two mechanisms, namely, fibrin bindingvia the kringle domains and the novel property of being converted toplasmin at the site of new thrombus formation by the action of one ofthe enzymes involved in generation of the thrombus and preferablylocalised there.

Factor Xa (E.C.3.4.21.6) is a serine protease which converts humanprothrombin to thrombin by specific cleavage of the Arg(273)-Thr(274)and Arg(322)-Ile(323) peptide bonds (Mann et al 1981, Methods inEnzymology 80 286-302). In human prothrombin, the Arg(273)-Thr(274) siteis preceded by the tripeptide Ile-Glu-Gly and the Arg(322)-Ile(323) siteis preceded by the tripeptide Ile-Asp-Gly. The structure required forrecognition by Factor Xa appears to be determined by the local aminoacid sequence preceding the cleavage site (Magnusson et al, 1975, in:"Proteases and Biological Control", 123-149, eds., Reich et al, ColdSpring Harbor Laboratory, New York). Specificity for the Ile-Glu-Gly-Argand Ile-Asp-Gly-Arg sequence is not absolute as Factor Xa has been foundto cleave other proteins, for example Factor VIII at positions 336, 372,1689 and 1721, where the preceding amino acid sequence differssignificantly from this format (Eaton et al, 1986 Biochemistry 25505-512). As the principal natural substrate for Factor Xa isprothrombin, preferred recognition sequences are those in which arginineand glycine occupy the P1 and P2 positions, respectively, an acidicresidue (aspartic or glutamic acid) occupies the P3 position andisoleucine or another small hydrophobic residue (such as alanine,valine, leucine or methionine) occupies the P4 position. However, asFactor Xa can cleave sequences which differ from this format, othersequences cleavable by Factor Xa may be used in the invention, as canother sequences cleavable by other enzymes of the clotting cascade.

Conversion of plasminogen to plasmin by tPA and uPA involves cleavage ofthe peptide bond between arginine 561 and valine 562 to produce adisulphide linked, two chain protein with an amino-terminal valine onthe light (protease domain) chain and a carboxy-terminal arginine on theheavy chain. Plasminogen is not cleaved and activated to any significantextent by thrombin or Factor Xa and in order to make plasminogenanalogues which are cleavable by these preferred enzymes, the cleavagesite Pro(559), Gly(560), Arg(561), Val(562) recognised by tPA and uPAhas to be altered. To make plasminogen analogues which are cleaved by,for example, Factor Xa, an amino acid sequence cleavable by Factor Xamay be substituted into the plasminogen molecule. The sequenceIle-Glu-Gly-Arg which is at one of the sites in prothrombin cleaved byFactor Xa may be such a sequence. Other possibilities would be sequencesor mimics of sequences cleaved by Factor Xa in other proteins orpeptides. A plasminogen analogue in which Pro(558) is removed andreplaced by Ile-Glu, may have the Arg(561)-Val(562) (wild-typeplasminogen numbering) peptide bond cleaved by Factor Xa to produce adisulphide-linked, two-chain plasmin analogue, with an amino-terminalvaline on the light (protease domain) chain and a carboxy-terminalarginine on the heavy chain. DNA coding for the Ile-Glu-Gly-Arg sequenceas the carboxy-terminal part of a cleavable linker as a proteinproduction aid is disclosed in UK Patent Application GB-A-2160206 butthe use of an Ile-Glu-Gly-Arg sequence to allow an altered activationprocess for a zymogen is not disclosed in that specification.

Cleavage and activation of plasminogen variants or other potentiallyactivatable proteinaceous compounds by an enzyme of the clotting cascadesuch as thrombin or Factor Xa can be measured in a number of ways, forexample by SDS-PAGE analysis, and in the case of plasminogen variants byassaying for the formation of plasmin using the S2251 chromogenic assayor a fibrin gel lysis assay.

Thrombin (E.C. 3.4.21.5) is a serine protease which catalyses theproteolysis of a number of proteins including fibrinogen (A alpha and Bbeta chains), Factor XIII, Factor V, Factor VII, Factor VIII, protein Cand antithrombin III. The structure required for recognition by thrombinappears to be partially determined by the local amino acid sequencearound the cleavage site but is also determined to a variable extent bysequence(s) remote from the cleavage site. For example, in thefibrinogen A alpha chain, residues P2 (Val), P9 (Phe) and P10 (Asp) arecrucial for α-thrombin-catalysed cleavage at the Arg(16)-Gly(17) peptidebond (Ni, F. et al 1989, Biochemistry 28 3082-3094). Comparative studiesof several proteins and peptides which are cleaved by thrombin has ledto the proposal that optimum cleavage sites for α-thrombin may have thestructure of (i) P4-P3-Pro-Arg-P1'-P2' where each of P3 and P4 isindependently a hydrophobic amino acid (such as valine) and each of P1'and P2' is independently a non-acidic amino acid such as a hydrophobicamino acid like valine, or (ii) P2-Arg-P1' where P2 or P1' is glycine(Chang, J. 1985, Eur. J. Biochem. 151 217-224). There are, however,exceptions to these general structures which are cleaved by thrombin andwhich may be used in the invention.

To produce a plasminogen analogue which could be cleaved and activatedby thrombin, a site recognised and cleaved by thrombin may besubstituted into the plasminogen molecule at an appropriate location. Anamino acid sequence such as that cleaved by thrombin in the fibrinogen Aalpha chain may be used. Other possible sequences would include thoseinvolved in the cleavage by thrombin of fibrinogen B beta, Factor XIII,Factor V, Factor VII, Factor VIII, protein C, anti-thrombin III andother proteins whose cleavage is catalysed by thrombin. An example of athrombin cleavable analogue of plasminogen may be one in which thesequence Pro(559), Gly(560) is changed to Gly(559), Pro(560) to producea sequence Gly(559)-Pro(560)-Arg(561)-Val(562) which is identical tothat found at positions 17-20 in fibrinogen A alpha. This is not theprincipal thrombin cleavage site in fibrinogen A alpha but thrombin cancleave the Arg(19)-Val(20) peptide bond.

Such subtle changes are important if the important features of fullactivity and stability are to be retained in the mutant derivative, asis preferred.

In a preferred embodiment the invention relates to plasminogen analogueswith single or multiple amino acid substitutions, additions or deletionsbetween residues Pro(555) and Cys(566) inclusive. Such plasminogenanalogues are cleaved by thrombin, Factor Xa or other enzymes involvedin blood clotting to produce plasmin analogues with fibrinolyticactivity.

Plasminogen analogues in accordance with the preferred embodiment of theinvention may contain other modifications (as compared to wild-typeglu-plasminogen) which may be one or more additions, deletions orsubstitutions. An example of such a modification would be the addition,removal, substitution or alteration of one or more kringle domains toenhance fibrin binding activity.

An example of a modification involving deletion would be lys-plasminogenvariants of plasminogen analogues in which the amino terminal 68, 77 or78 amino acids have been deleted. Such variants may have enhanced fibrinbinding activity as has been observed for lys-plasminogen compared towild-type glu-plasminogen (Bok, R. A. and Mangel, W. F. 1985,Biochemistry 24 3279-3286).

The plasmin inhibitor alpha-2 antiplasmin is present in the blood andbecomes incorporated into the fibrin matrix of blood clots. The role ofthis inhibitor is to restrict plasmin activity in the clot and in thecirculation. For the highly clot selective analogues of plasminogen ofthe present invention it may be advantageous to introduce a mutation inthe serine protease domain that interferes with plasmin inhibitorbinding. This mutation could be in a position analogous to that shown toprevent inhibitor binding to tissue plasminogen activator (Madison, E.L. et al 1989 Nature 339 721-724).

Other plurally-modified plasminogen analogues in accordance with theinvention may include one or more modifications to prevent, reduce oralter glycosylation patterns. Plasminogen analogues incorporating suchmodifications may have a longer half-life, reduced plasma clearanceand/or higher specific activity.

Other proteins may also be altered so that they are cleaved or otherwiseactivated, by enzymes involved in blood clotting, to be fibrinolyticallyactive or to be inhibitory of clot formation. Single chain urokinaseplasminogen activator (scuPA) is an example of a fibrinolytic proteinand protein C is an example of an enzyme involved in inhibition of bloodclotting which could be so activated.

scuPA is activated to two chain uPA (tcuPA) by plasmin cleavage of theLys(158)-Ile(159) peptide bond. Thrombin inactivates scuPA by cleavingthe Arg(156)-Phe(157) peptide bond. An analogue of scuPA could beconstructed in which the amino acid sequence around the cleavage sitewas altered so that cleavage by thrombin, or another enzyme involved inblood clotting, would produce active tcuPA.

Protein C is cleaved to its activated form by the action of thrombinbound to thrombomodulin. A protein C analogue within the scope of thisinvention is modified so as to be cleavable by thrombin per se to formactivated protein C.

Fusion proteins may be constructed to achieve selective release offibrinolytic or anticoagulant proteins at the site of blood clotting. Toachieve this, proteins involved in fibrinolysis or inhibition ofcoagulation are joined by a linker region which is cleavable by anenzyme involved in blood clotting. Examples of proteins which may beincorporated into such a cleavable protein include tPA, uPA,streptokinase, plasminogen, protein C, hirudin and anti-thrombin III.Fusion of such proteins to a protein with a favourable property notdirectly related to dissolution of blood clots (for example albumin,which has a long plasma half-life) may also be beneficial.

Preferred features of plasminogen analogues within the scope of theinvention also apply, where appropriate, to other compounds of theinvention, mutatis mutandis.

Compounds in accordance with the first aspect of the invention can besynthesised by any convenient route. According to a second aspect of theinvention there is provided a process for the preparation of aproteinaceous compound as described above, the process comprisingcoupling successive amino acid residues together and/or ligatingoligopeptides. Although proteins may in principle be synthesised whollyor partly by chemical means, the route of choice will be ribosomaltranslation, preferably in vivo, of a corresponding nucleic acidsequence. The protein may be glycosylated appropriately.

It is preferred to produce proteins in accordance with the invention byusing recombinant DNA technology, particularly when they are analogues(whether by amino acid substitution, deletion or addition) of naturalproteins. DNA encoding plasminogen or another natural protein may befrom a cDNA or genomic clone or may be synthesised. Amino acidsubstitutions, additions or deletions are preferrably introduced bysite-specific mutagenesis. Suitable DNA sequences encodingglu-plasminogen, lys-plasminogen and plasminogen analogues and othercompounds within the scope of the invention may be obtained byprocedures familiar to those having ordinary skill in geneticengineering. For several proteins, including for example tissueplasminogen activator, it is a routine procedure to obtain recombinantprotein by inserting the coding sequence into an expression vector andtransfecting the vector into a suitable host cell. A suitable host maybe a bacterium such as E. coli, a eukaryotic microorganism such as yeastor a higher eukaryotic cell. Plasminogen, however, is unusuallydifficult to express and several unsuccessful attempts have been made atproducing recombinant plasminogen in mammalian cells (Busby S. et al1988, Fibrinolysis 2, Suppl. 1, 64; Whitefleet-Smith et al, 1989, Arch.Bioc. Biop. 271 390-399). It may be possible to express plasminogen inE. coli but the protein would be made in an insoluble form and wouldhave to be renatured. Satisfactory renaturation would be difficult withcurrent technology. Plasminogen has been expressed in insect cells usinga baculovirus vector-infected cell system at levels of 0.7-1.0 μg/10⁶cells (measured 66 hours post infection) (Whitefleet-Smith et al, ibid)but this method does not generate a stable cell line producingplasminogen and any post-translational modifications, such asglycosylation, may not be authentic.

According to a third aspect of the invention, there is providedsynthetic or recombinant nucleic acid coding for a proteinaceouscompound as described above. The nucleic acid may be RNA or DNA.Preferred characteristics of this aspect of the invention are as for thefirst aspect.

According to a fourth aspect of the invention, there is provided aprocess for the preparation of nucleic acid in accordance with the thirdaspect, the process comprising coupling successive nucleotides togetherand/or ligating oligo- and/or poly-nucleotides.

Recombinant nucleic acid in accordance with the third aspect of theinvention may be in the form of a vector, which may for example be aplasmid, cosmid or phage. The vector may be adapted to transfect ortransform prokaryotic (for example bacterial) cells and/or eukaryotic(for example yeast or mammalian) cells. A vector will comprise a cloningsite and usually at least one marker gene. An expression vector willhave a promoter operatively linked to the sequence to be inserted in thecloning site, and, preferably, a sequence enabling the protein productto be secreted. Expression vectors and cloning vectors (which need notbe capable of expression) are included in the scope of the invention.

Certain vectors are particularly useful in the present invention.According to a fifth aspect of the invention, there is provided a vectorcomprising a first nucleic acid sequence coding for a protein orembodying a cloning site, operatively linked to a second nucleic acidsequence containing a strong promoter and enhancer sequence derived fromhuman cytomegalovirus, a third nucleic acid sequence encoding apolyadenylation sequence derived from SV40 and a fourth nucleic acidsequence coding for a selectable marker expressed from an SV40 promoterand having an additional SV40 polyadenylation signal at the 3' end ofthe selectable marker sequence.

It is to be understood that the term "vector" is used in thisspecification in a functional sense and is not to be construed asnecessarily being limited to a single nucleic acid molecule. So, forexample, the first, second and third sequences of the vector definedabove may be embodied in a first nucleic acid molecule and the fourthsequence may be embodied in a second nucleic acid molecule.

The selectable marker may be any suitable marker. The gpt marker isappropriate.

Such a vector enables the expression of such proteins as plasminogen andplasminogen analogues (including glu-plasminogen and lys-plasminogen)which may otherwise be difficult to express.

This aspect of the invention provides the construction of a vector whichis useful for the expression of foreign genes and cDNAs and for theproduction of heterologous proteins in mammalian cells. The particularembodiment exemplified is the construction of stable cell lines whichare capable of expressing plasminogen and plasminogen analogues at highlevels.

Using a vector, for example as described above, heterologous proteins,such as plasminogen and plasminogen analogues, are preferably expressedand secreted into the cell culture medium in a biologically active formwithout the need for any additional biological or chemical procedures.Suitable cells or cell lines to be transformed are preferably mammaliancells which grow in continuous culture and which can be transfected orotherwise transformed by standard techniques. Examples of suitable cellsinclude Chinese hamster ovary (CHO) cells, mouse myeloma cell lines suchas P3X63-Ag8.653, COS cells, HeLa cells, BHK cells, melanoma cell linessuch as the Bowes cell line, mouse L cells, human hepatoma cell linessuch as Hep G2, mouse fibroblasts and mouse NIH 3T3 cells.

It appears that the use of CHO cells as hosts for the expression ofplasminogen and plasminogen analogues is particularly beneficial.According to a sixth aspect of the invention, there is thereforeprovided a chinese hamster ovary (CHO) cell transformed to expressplasminogen or a plasminogen analogue.

CHO or other cells, such as yeast (for example Saccharomyces cerevisiae)or bacteria (for example Escherichia coli) may be preferred for theexpression of other proteinaceous compounds of the invention. Accordingto a seventh aspect of the invention, there is provided a cell or cellline transformed by nucleic acid and/or a vector as described above.Transformation may be achieved by any convenient method; electroporationis a method of choice.

Proteinaceous compounds of the present invention may be used withinpharmaceutical compositions for the prevention or treatment ofthrombosis or other conditions where it is desired to produce localfibrinolytic and/or anticoagulant activity. Such conditions includemyocardial and cerebral infarction, arterial and venous thrombosis,thromboembolism, post-surgical adhesions, thrombophlebitis and diabeticvasculopathies.

According to an eighth aspect of the invention, there is provided apharmaceutical composition comprising one or more compounds inaccordance with the first aspect of the invention and a pharmaceuticallyor veterinarily acceptable carrier. Such a composition may be adaptedfor intravenous administration and may thus be sterile. Examples ofcompositions in accordance with the invention include preparations ofsterile plasminogen analogue(s) in isotonic physiological saline and/orbuffer. The composition may include a local anaesthetic to alleviate thepain of injection. Compounds of the invention may be supplied in unitdosage form, for example as a dry powder or water-free concentrate in ahermetically sealed container such as an ampoule or sachet indicatingthe quantity of protein. Where a compound is to be administered byinfusion, it may be dispensed by means of an infusion bottle containingsterile water for injections or saline or a suitable buffer. Where it isto be administered by injections, it may be dispensed with an ampoule ofwater for injection, saline or a suitable buffer. The infusible orinjectable composition may be made up by mixing the ingredients prior toadministration. Where it is to be administered as a topical treatment,it may be dispensed in a suitable base.

The quantity of material to be administered will depend on the amount offibrinolysis or inhibition of clotting required, the required speed ofaction, the seriousness of the thromboembolic position and the size ofthe clot. The precise dose to be administered will, because of the verynature of the condition which compounds of the invention are intended totreat, be determined by the physician. As a guideline, however, apatient being treated for a mature thrombus will generally receive adaily dose of a plasminogen analogue of from 0.01 to 10 mg/kg of bodyweight either by injection in for example up to 5 doses or by infusion.

The invention may be used in a method for the treatment or prophylaxisof thrombosis, comprising the administration of an effective non-toxicamount of a compound in accordance with the first aspect. According to afurther aspect of the invention, there is therefore provided the use ofa compound as described above in the preparation of a thrombolyticand/or anticoagulant agent.

The invention concerns especially the DNAs, the vectors, the transformedhost strains, the plasminogen analogue proteins and the process for thepreparation thereof as described in the examples.

The following figures and examples of the invention are offered by wayof illustration, and not by way of limitation. Examples 1 to 3 describethe expression vector used for the expression of plasminogen andplasminogen variants from higher eukaryotic cells. Subsequent examplesdescribe the expression of plasminogen and plasminogen variants andtheir properties. In the drawings referred to in the examples:

FIG. 1 shows the construction of pGWH;

FIG. 2 shows the nucleotide sequence of the glu-plasminogen cDNA and thepredicted amino acid sequence;

FIG. 3 shows a map of the expression vector pGWHgP1;

FIG. 4 shows the cleavage site sequences of Factor Xa activatedplasminogen analogues;

FIG. 5 shows the cleavage site sequences of thrombin activatedplasminogen analogues;

FIG. 6 shows activation of X2 by Factor Xa and T2 by thrombin on afibrin agar gel;

FIG. 7 shows the activation of plasminogen mutants X3, T13 and T19 byfactor Xa (for X3) or thrombin (for T13 and T19); X3 is the subject ofExamples 5 and 21, T13 is the subject of Examples 13 and 24 and T19 isthe subject of Examples 16 and 26;

FIG. 8 shows the activation of plasminogen mutant T19 (Examples 16 and26) by thrombin, as determined by assay of plasmin;

FIG. 9 shows an SDS-PAGE gel showing cleavage of X2 by Factor Xa and T2by thrombin; and

FIG. 10 shows the rate of cleavage of plasminogen mutant T19 (Examples16 and 26) with thrombin.

EXAMPLE 1

The plasmid pSS1 is a signal sequence vector which provides a secretionsignal for any gene lacking such a sequence. pGW1 is derived from thisvector and pGWH is an expression vector containing a promoter.

Construction of pSS1

1. The plasmid pUC18 (FIG. 1.1) was used as the backbone of the vectoras it contains both an E. coli origin of replication, which allowsproduction of the plasmid DNA in E. coli and an ampicillin resistancegene, allowing selection for the plasmid in E. coli (FIG. 1.1). (pUC18is disclosed in Gene 19 259-268 (1982) and Gene 26 101-106 (1983) and isdeposited at the American Type Culture Collection under deposit no. ATCC37253.) pUC18 also contains polylinker into which the synthetic DNA wasinserted but this polylinker has an EcoRI site which it was necessary todelete before insertion of the synthetic sequence. This was done bycleaving the DNA with EcoRI and treating with mung bean nuclease, asingle stranded nuclease, and then religating the plasmid DNA (FIG.1.2).

2. The modified pUC18 DNA was cleaved with HindIII and BamHI and intothese sites a synthetic fragment of DNA:(5'AGCTTCCACCATGAAGTGCTCCTGGGTGATCTTCTTCCTGATGGCCGTGGTGACCGGCGTGAACTCGCGAGATCTAGAGTCGACCTGCAGGATATCGAATTCATT 3' (top strand,SEQ ID NO:41), 5'GATCAATGAATTCGATATCCTGCAGGTCGACTCTAGATCTCGCGAGTTCACGCCGGTCACCACGGCCATCAGGAAGAAGATCACCCAGGAGCACTTCATGGTGGA 3' (bottom strand,SEQ ID NO:42)) containing an immunoglobulin signal sequence (Nature,331, 173-175 Rogelj et al, 1988) plus a polylinker, which contains avariety of restriction enzyme sites, and also a 237 base pair BclI-BamHIfragment, isolated from SV40 DNA and which contains a polyadenylationsignal, were ligated in a three way reaction (FIG. 1.3). Polyadenylationsignals from other genes, such as bovine growth hormone, could also beused in the construction of this vector. Remnants of the pUC18 backbone,namely the KpnI and SmaI sites, remained in this construct and so thesesites were deleted by digestion of the plasmid DNA with KpnI and BamHI,removal of the fragment and insertion of a bottom strand linker(5'GATCCGTAC 3') which destroys the KpnI and SmaI sites but reforms theBamHI site (FIG. 1.3).

3. In order to make this vector useful for transient expression in COScells a synthetic 90 base pair SV40 origin of replication (5'TATGAAGACGTCGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGGG 3' (top strand, SEQ ID NO:43)),5'CGCCCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCGACGTCTTCA 3' (bottom strand, SEQ ID NO:44)was ligated into the NdeI-NarI sites of pUC18 to replace a 53 base pairfragment (FIG. 1.4).

4. A synthetic DNA sequence (5'AAGCGGCCGCGGCCATGCCGGCCACTAGTCTCGAGTT 3'(top strand, SEQ ID NO:45); 5'AACTCGAGACTAGTGGCCGGCATGGCCGCGGCCGCTT 3'(bottom strand, SEQ ID NO:46)), which encodes restriction enzyme siteswhich cut infrequently in the mammalian genome and which aidslinearization of the plasmid DNA before transfection, was ligated intothe plasmid at the SspI site to form the promoter-less vector pSS1 (FIG.1.5).

5. The nucleotide sequence of the entire plasmid was confirmed.

Construction of pGW1

Many cDNAs or genes to be expressed already have a signal sequence andso pSS1 was modified to remove the secretion signal.

6. The DNA was cleaved with HindIII and NruI, the fragment removed, anda linker (5'AGCTTCCCGGGATAGGTACCTCG 3' (top strand, SEQ ID NO:66),5'CGAGGTACCTATCCCGGGA 3' (bottom strand, SEQ ID NO:67)) containing theHindIII, SmaI, KpnI and NruI sites was inserted (FIG. 1.6). In additionto removing the signal sequence this also adds two restriction enzymesites to the polylinker thus making it more versatile. This promoterlessvector is called pGWI and its correct assembly was confirmed bynucleotide sequence analysis of the entire plasmid.

Construction of pGWH

7. The plasmid pSS1 has no promotor or enhancer sequence. This can beconveniently added by ligating appropriate fragments of DNA into thepolylinker, for example at the HindIII site. One promotor/enhancersequence suitable for use is the immediate early transcriptionalregulatory region of human cytomegalovirus (HCMV) (Proc. Natl. Acad.Sci. USA, 81, 659-663, Thomsen et al, 1984), although other regulatoryregions could be used e.g. Rous Sarcoma Virus long terminal repeat (RSVLTR), SV40 early or late promoter/enhancer region, Mouse mammary tumourvirus (MMTV) LTR, mouse metallothionein promoter. This was inserted intopGW1 at the HindIII site and then the orientation was checked byrestriction endonuclease digestion. The 5' Hind III site was thendeleted by performing a partial digestion with Hind III, such that onlythe 5' site was cleaved. This site was then removed by treatment withmung bean nuclease and subsequent religation to form pGWH (FIG. 1.7).The correct assembly of the vector was confirmed by nucleotide sequenceanalysis of the entire plasmid.

8. A DNA fragment including the selectable marker gene gpt and the SV40early promoter/enhancer sequence and polyadenylation sequence was clonedinto the BamHI site of the vector to form pGWHg, and allows selection ofcells which have stably integrated the plasmid DNA. Genes encodingproteins conferring resistance to G418 or hygromycin, or a variety ofmetabolic selections, could also be used.

This particular expression system is preferred because of its efficiencybut its use is not intended to limit the scope of the present invention.In the literature there are described many alternative methods ofexpressing genes in mammalian cells and such expression systems are wellknown to those skilled in the art of genetic engineering and have beenat least partially documented by Gorman in "DNA Cloning Vol. II: APractical Approach" (D. M. Glover, ed. IRL Press, Oxford (1985) pp143-190).

Example 2--Expression of Glu-Plasminogen

Methods that can be used for the isolation of cDNA are well documentedand a procedure that has been used for the isolation of plasminogen cDNAis summarised in the following protocol. The human plasminogen cDNA hasbeen cloned and sequenced (Forsgren et al, FEBS Letters, 213, 254-260(1987))

1. The RNA was prepared from fresh human liver using the guanidinethiocyanate method (Chirgwin et al Biochemistry 10:5294 (1979)) andpurified using an oligo-dT column (Aviv and Leder PNAS 69:1408 (1972))

2. The cDNA library was prepared as described in the Amersham Protocol("cDNA Synthesis and Cloning System", Amersham International plc, 1985).The double stranded cDNA was ligated into a lambda vector.

3. Plaques were screened for plasminogen cDNA by hybridization tonitrocellulose replicates using ³² P-labelled oligonucleotide probes (17mers), representing the 3' and 5' ends of plasminogen, in a buffercontaining 6×SSC (SSC is 150 mM NaCl, 15 mM sodium citrate),5×Denhardt's, 0.2% SDS and 0.1 mg/ml salmon sperm DNA at roomtemperature overnight. Filters were washed using 6×SSC, 0.1% SDS at 47°C. Positive plaques were purified, subjected to plasmid rescue and thepackaged recombinant plasmid clones or their subclones were sequenced bya modification of the dideoxy method using dATP-5'-α-[35_(S) ]thiophosphate (see Methods section). This cDNA encodes a glu-plasminogenprotein of 791 amino acids which corresponds with the length ofplasminogen reported by Forsgren et al, (ibid) and contains an extraamino acid (Ile65) when compared to the amino acid sequence determinedby protein sequencing (Sottrup-Jensen et al, ibid). The nucleotidesequence of the cDNA and the 791 amino acid sequence of glu-plasminogenis shown in FIG. 2.

Other methods of isolation can be used, for example mRNA isolated fromcells which produce plasminogen can be prepared using the guanidinethiocyanate method (Chirgwin et al Biochemistry 10:5294 (1979)) and acomplementary first strand of DNA synthesized using reversetranscriptase. The Polymerase Chain Reaction (PCR) can then be used toamplify the plasminogen sequence (Saiki R. et al, Science, 239, 487-491(1988)). The PCR reaction could also be used to amplify the sequencefrom DNA prepared from a genomic or cDNA library which containssequences encoding plasminogen. Alternatively, the gene could beassembled from chemically synthesised oligonucleotides.

The 2.5 kb BalI-SphI glu-plasminogen fragment was sub-cloned into thepolylinker of pUC18 at the SmaI-SphI sites (FIG. 2). The plasminogencDNA was then cleaved out of pUC18 on a KpnI-SphI fragment and ligatedinto the vector pGWH to create pGWHP, prepared as described in Example1, at the KpnI and EcoRI sites using an EcoRI-SphI linker (5'AATTCCATG3'). Thus transcription through the plasminogen cDNA can initiate at theHCMV promoter/enhancer (FIG. 3). The selectable marker gpt, expressedfrom the SV40 promoter and with a polyadenylation signal at its 3' end,was cloned into the BamHI site of pGWHP to create pGWHgP1 (FIG. 3) andthe orientation checked by restriction enzyme nuclease digestion.Plasmid DNA was introduced into CHO cells by electroporation using 800 Vand 25 μF as described in the methods section below. Selective medium(250 μl/ml xanthine, 5 μg/ml mycophenolic acid, 1×hypoxanthine-thymidine (HT)) was added to the cells 24 hours posttransfection and the media changed every two to three days. Platesyielding gpt-resistant colonies were screened for plasminogen productionusing an ELISA assay. Cells producing the highest levels of antigen werere-cloned and the best producers scaled up into flasks with productionbeing carefully monitored. Frozen stocks of all these cell lines werelaid down. The cell lines C1.44 and C1.75, which both produceglu-plasminogen at a concentration of >3 mg/liter, were scaled up intoroller bottles to provide conditioned medium from which plasminogenprotein was purified using lysine SEPHAROSE 4B. (The word SEPHAROSE is atrade mark.) The purified plasminogen was then assayed for its abilityto be cleaved to plasmin by tPA or streptokinase using the fibrin agarclot assay. Cleavage of the zymogen was also established using SDS PAGE(Nature, 227, 680, Laemmli, 1970).

The techniques of genetic manipulation, expression and proteinpurification used in the manufacture of this wild type plasminogen, aswell as those of the modified plasminogen examples to follow, are wellknown to those skilled in the art of genetic engineering. A descriptionof most of the techniques can be found in one of the followinglaboratory manuals: "Molecular Cloning" by T. Maniatis, E. F. Fritschand J. Sambrook published by Cold Spring Harbor Laboratory, Box 100, NewYork, or "Basic Methods in Molecular Biology" by L. G. Davis, M. D.Dibner and J. F. Battey published by Elsevier Science publishing Co Inc,New York.

Additional and modified methodologies are detailed in the methodssection below.

Example 3--Construction and Expression of X1

Plasminogen analogues which are altered around the Arg(561), Val(562)cleavage sites have been constructed in order to modify the site andallow recognition and cleavage by alternative enzymes. X1 is aplasminogen analogue in which the amino acid residue Pro(559) isreplaced by Ile and Glu (FIG. 4). This site was based on a Factor Xacleavage site in prothrombin. The modification strategy in this examplewas to sub-clone the 1.87 Kb KpnI-HincII fragment, from the plasminogencDNA in a pUC18 vector, into the single stranded bacteriophage M13mp18to facilitate the mutagenesis. Single strand template was prepared andthe mutations made by oligonucleotide directed mismatch mutagenesis. Inthis case a 21 base long oligonucleotide (5'CCCTTCCCTCGATACATTTCT 3',SEQ ID NO:47) was used to direct the mutagenesis. Clones carrying themutation were identified by sequencing and then fully sequenced toensure that no other mutation had inadvertently been introduced.Replicative form (RF) DNA was then prepared and the mutation transferredinto the expression vector containing the Glu plasminogen (as describedin Example 2) by replacing the wild type KpnI-EcoRV fragment with themutated fragment. The pGWHg plasmid carrying the mutant plasminogen wasthen linearized with the restriction endonuclease NotI and introducedinto CHO cells by electroporation. The expression protocol was then thesame as that described in Example 2. The cell line used to produce thismutant protein is C7.9. Activation and cleavage of this mutant withpurified Factor Xa was investigated as described for Examples 20 and 29.

Example 4--Construction and Expression of X2

The procedure of Example 3 was generally followed except that the primerused was the 22 mer (5'CCTTCCCTCGATGCCACATTTC 3', SEQ ID NO:48). Theresulting mutant derivative of plasminogen has the following amino acidchanges: Pro(559) to Gly, Gly(560) to Ile and addition of Glu and Glybefore Arg(561) (FIG. 4). This cleavage site is based on a Factor Xacleavage site in prothrombin. The cell line C8.24 was scaled up toproduce this mutant protein. Otherwise, the procedure of Example 3 wasgenerally followed. Activation and cleavage of this mutant wasinvestigated as described in Examples 20 and 27.

Example 5--Construction and Expression of X3

In X3, Pro(559) has been substituted by Gly, Ala, Ile and Glu using the48 mer (5'CCCCCCCACAACCCTTCCCTCTATTGCACCACATTTCTTCGGCTCCAC 3', SEQ IDNO:49) (FIG. 4). The cell line C37.4 has been used to produce thisprotein which has a cleavage site based on a Factor Xa cleavage site inprothrombin. Otherwise, the procedure of Example 3 was generallyfollowed. Activation of this mutant is described in Example 21 below.

Example 6--Construction and Expression of X5

X5 has Pro(559) replaced by Gly, Tyr, Ile and Asp using a 48 mer(5'CCCCCCCACAACCCTTCCGTCTATGTAACCACATTTCTTCGGCTCCAC 3', SEQ ID NO:50)(FIG. 4). The cell line C39.7 has been used to produce this proteinwhich has a cleavage site based on a Factor Xa cleavage site inprothrombin. Otherwise, the procedure of Example 3 was generallyfollowed. Activation of this mutant is described in Example 21 below.

Example 7--Construction and Expression of X6

In addition to the mutation in X5, X6 has Val(561) replaced by Ile (FIG.4). This was made using the 52 mer(5'CACACCCCCCCACAATCCTTCCGTCTATGTAACCACATTTCTTCGGCTCCAC 3', SEQ IDNO:51). The cell line C36.1 has been used to produce this protein.Otherwise, the procedure of Example 3 was generally followed. Activationof this mutant is described in Example 21 below.

Example 8--Construction and Expression of T1

T1 is a plasminogen derivative in which Pro(559) and Gly(560) have beeninterchanged to give Gly at position 559 and Pro at 560 (FIG. 5). Thiscleavage site mimics the thrombin cleavage site at Arg(19)-Val(20) inthe fibrinogen A alpha chain. The procedure of Example 3 was generallyfollowed except that the primer used was the 21 mer(5'CAACCCTTGGACCACATTTCT 3', SEQ ID NO:52). The cell line producing theT1 mutant is C6.23. Activation and cleavage of this protein aredescribed in Examples 22 and 28 below.

Example 9--Construction and Expression of T2

T2 is a plasminogen derivative which has been modified from wild typeplasminogen in the same way as T1 but an extra Gly amino acid has beenadded between Gly(559) and Pro(560) (FIG. 5). The procedure of Example 3was generally followed except that the primer used to make this mutantis a 22 mer (5'ACCCTTGGACCACCACATTTCT 3', SEQ ID NO:53). The cell lineC5.16 was used to produce this mutant protein. Activation and cleavageof this mutant are shown in Examples 22 and 28 below.

Example 10--Construction of T6

In the T6 protein there are two sites of amino acid change. The aminoacids Pro(559), Gly(560), Arg(561), Val (562) have been replaced by sixamino acids to become Gly(559), Val(560), Val(561), Pro(562), Arg(563),Gly(564). In addition to these changes, Val(553), Lys(556), Lys(567)have been replaced by Leu, Glu and Leu respectively using a 61 mer(5'GGGCCACACACCCCCCCACTCCCCTAGGCACAACTCCACATAGCTCCGGCTCCAGTTGAGG 3', SEQID NO:54) (FIG. 5). This modification is based on a thrombin cleavagesite in Factor XIII. The cell line C45.1 was used to produce thisprotein. Otherwise, the procedure of Example 3 was generally followed.Activation and cleavage of this protein is described in Examples 23 and29 below.

Example 11--Construction and Expression of T7

In another modification based on a thrombin cleavage site in FactorXIII, T7 incorporates the first set of changes described for T6 namelythe replacement of Pro(559), Gly(560), Arg(561), Val(562) by six aminoacids to become Gly(559), Val(560), Val(561), Pro(562), Arg(563),Gly(564). In addition Val(553), Lys(556) and Lys(557) have been replacedby Leu, Gln and Leu respectively using the 60 mer(5'GGCCACACACCCCCCCACTCCCCTAGGCACAACTCCACATAGTTGCGGCTCCAGTTGAGG 3', SEQID NO:55) (FIG. 5). The cell line C26.5 was used to produce thisprotein. Otherwise, the procedure of Example 3 was generally followed.Activation and cleavage of this protein is described in Examples 23 and29 below.

Example 12--Construction and Expression of T8

T8 is based on the thrombin cleavage site in Factor XIII and in thisprotein Pro(559), Gly(560), Arg(561), Val(562) have been replaced byVal, Glu, Leu, Gln, Gly, Val, Val, Pro, Arg, Gly using a 61 mer(5'CACACACCCCCCCACTCCCCTAGGCACTACTCCTTGTAGTTCTACACATTTCTTCGGCTCC 3', SEQID NO:56) (FIG. 5). The cell line C34.5 has been used to produce thisprotein. Otherwise, the procedure of Example 3 was generally followed.Activation and cleavage of this protein is described in Examples 23 and29 below.

Example 13--Construction and Expression of T13

In the plasminogen derivative T13 the two amino acids Pro(559),Gly(560), have been replaced by three amino acids Val, Val and Pro usinga 41 mer (5'CACCCCCCCACAACCCTAGGTACAACACATTTCTTCGGCTC 3', SEQ ID NO:57)(FIG. 5). The cell line C51.1 was used to produce this protein.Otherwise, the procedure of Example 3 was generally followed. Activationand cleavage of this protein is described in Examples 24 and 29 below.

Example 14--Construction and Expression of T14

The plasminogen analogue T14 has a thrombin cleavage site based on asite cleaved by thrombin in calcitonin. In this mutant the amino acidsGly and Tyr are inserted between Cys(558) and Pro(559) and in additionGly(560) is deleted (FIG. 5). These mutations were made using a 41 mer(5'CACCCCCCCACAACCCTAGGGTATCCACATTTCTTCGGCT 3', SEQ ID NO:58). The cellline used to produce this protein was C61.1. Otherwise, the procedure ofExample 3 was generally followed.

Example 15--Construction and Expression of T17

The protein T17 has a cleavage site based on a site cleaved by thrombinin cholecystokinin. This protein has Ser inserted between Pro559 and Gly560 and was made using a 38 mer(5'CACCCCCCCACAACCCTTCCACTAGGACATTTCTTCGG 3', SEQ ID NO:59) (FIG. 5).The cell line C49.7 was used to produce this protein. Otherwise, theprocedure of Example 3 was generally followed. Activation and cleavageof this protein is described in Examples 25 and 29 below.

Example 16--Construction and Expression of T19

The cleavage site of this protein is based on a thrombin cleavage sitein factor XIII. This mutant differs from T8 in that the P1' amino acidis Val rather than Gly. Cleavage produces two chain T19 plasmin with anative light chain sequence. In this protein Pro(559), Gly(560),Arg(561) have been replaced by Val, Glu, Leu, Gln, Gly, Val, Val, Pro,Arg using a 61 mer(5'CACACCCCCCCACAACCCTTGGGACTACTCCCTGCAATTCTACACATTTCTTCGGCTCCAC 3', SEQID NO:60) (FIG. 5). The cell line, C53.5, was used to produce theprotein. Otherwise, the procedure of Example 3 was generally followed.The activation and cleavage analysis of this protein is presented inExamples 26 and 29 below.

Example 17--Construction and Expression of T20

The cleavage site of this protein is similar to T19 but the aminoterminal sequence of the plasmin light chain generated by cleavage hasVal(562), Val(563) deleted. In this protein Pro(559), Gly(560),Arg(561), Val(562) and Val(563) have been replaced by Val, Glu, Leu,Gln, Gly, Val, Val, Pro, Arg using a 58 mer(5'GGCCACACACCCCCCCCTTGGGACTACTCCCTGCAATTCTACACATTTCTTCGGCTCC 3', SEQ IDNO:61) (FIG. 5). The cell line C54.2 was used to produce protein.Otherwise, the procedure of Example 3 was generally followed.

Example 18--Construction and Expression of T21

This mutant differs from T6 in that the P1' amino acid is Val ratherthan Gly. Cleavage produces two chain T21 plasmin with a native lightchain sequence. The cDNA encoding this protein was made using the T6cDNA template, described in Example 10, and the 23 mer(5'CACCCCCCCACTACCCTAGGCAC 3', SEQ ID NO:62) (FIG. 5). The cell lineC55.9 has been used to produce this protein. Otherwise, the procedure ofExample 3 was generally followed.

Example 19--Construction and Expression of T22

This mutant differs from T7 in that the P1' amino acid is Val ratherthan Gly. Cleavage produces two chain T22 plasmin with a native lightchain sequence (FIG. 5). The cDNA encoding this protein was made in a T7cDNA background, as described in Example 11, using the 23 mer describedfor T21. The cell line C56.11 has been used to produce this protein.Otherwise, the procedure of Example 3 was generally followed.

Example 20--Activation of X1 and X2

Activation of the X1 and X2 proteins to plasmin by Factor Xa was testedusing a fibrin lysis assay. Generation of plasmin is detected by theappearance of a zone of clearance developing in a fibrin-agarose gel asdescribed in Method 12.1 (see Methods section below). 25 μl lots ofpurified plasminogen mutant (635 μg/ml) were incubated with 2.5 μlpurified Factor Xa (0.35 μg) at 37° C. Generation of plasmin was assayedby adding 10 μl samples from the incubation to wells in a fibrin agargel. Samples of plasminogen mutant incubated with Factor Xa gave a zoneof clearance on the gel which was not present in control samples whichhad not been incubated with Factor Xa. Activation of X2 to plasmin byFactor Xa is shown in FIG. 6.

Example 21--Activation of X3, X5 and X6

Purified X3 protein was assayed for activation using the linkedchromogenic assay (see Method 12.3). Results of this assay are shown inFIG. 7 in which the increase in absorbance at 405 nm with timedemonstrates that plasmin activity is generated upon incubation of X3with Factor Xa. Similarly, X5 and X6 were shown to be activated uponincubation with Factor Xa.

Example 22--Activation of T1 and T2

The purified mutant proteins T1 and T2 were assayed for activation asdescribed in Example 20 except that the mutant proteins werepreincubated with thrombin (2551 plasminogen mutant (120 μg/ml) wasincubated with 2.5 μl thrombin (0.69 units)) and the wells in the fibringel were pretreated with hirudin to inhibit any activating effect of thethrombin which was used to make the gel. Both mutants were activated bythrombin as samples incubated with thrombin produced a zone of clearanceon the gel. Zones of clearance were not produced by control sampleswhich had not been incubated with thrombin. The results for T2 are shownin FIG. 6.

Example 23--Activation of T6, T7 and T8

The mutant proteins were assayed for activation using the linkedchromogenic assay (see Method 12.3). This assay demonstrated that T6, T7and T8 are not activated by thrombin (although they are cleaved--seeExample 29).

Example 24--Activation of T13

Purified T13 protein was assayed using the linked chromogenic assay asdescribed in Example 23. Results of this assay are shown in FIG. 7 inwhich the increase in absorbance at 405 nm with time demonstrates thatT13 is activated by thrombin. Activation was also detected using thedirect chromogenic assay as described in Example 26.

Example 25--Activation of T17

Purified T17 protein was assayed using the linked chromogenic assay asdescribed in Example 23. This assay demonstrated that thrombin activatesT17.

Example 26--Activation of T19

Purified T19 protein was assayed using the linked chromogenic assay asdescribed in Example 23. Results of this assay are shown in FIG. 7 inwhich the increase in absorbance at 405 nm with time demonstrates thatT19 is activated by thrombin.

The mutant protein T19 was also analysed using a direct chromogenicassay which allows quantitation of plasmin generated by activation (seeMethod 12.2). Results of this assay are shown in FIG. 8 in which thegeneration of plasmin with time demonstrates that T19 is activated bythrombin.

Example 27--Cleavage of X Mutants

Samples of 25 μg of X plasminogen mutants were incubated with 1.5 μgFactor Xa in 0.25 ml buffer and cleavage analysis was performed asdescribed in Method 11. FIG. 9 shows that the X2 plasminogen band atapproximately 92 kDa was cleaved to form a heavy chain plasmin band atapproximately 66 kDa. This indicates that the mutant amino acid sequencethat we have introduced is cleaved by Factor Xa and that the activationdemonstrated for X2 in Example 20 is a result of cleavage of theplasminogen analogue to produce plasmin.

Example 28--Cleavage of T1 and T2

Cleavage analyses of the purified proteins T1 and T2 were performed asdescribed in Example 27 except that thrombin (1.5 μg) was used insteadof Factor Xa. Cleavage of T2 to plasmin by thrombin is shown in FIG. 9thus confirming that the activation demonstrated in Example 24 is aresult of thrombin cleavage.

Example 29--Cleavage of T6, T7, T8, T13, T17 and T19

Samples of 12.5 μg plasminogen mutant were incubated with 2.8 μgthrombin as described in Method 11. The time course of cleavage of theplasminogen mutants was determined by quantitative gel scanning and thetimes for 50% cleavage of T6, T7, T8, T13, and T19 were 13, 40, 15, 70and less than 10 minutes respectively while the cleavage time for T17was approximately 30 hours. Gel scan data for cleavage of T19(disappearance of the plasminogen band) are shown in FIG. 10.

Example 30--Construction of Lys-3

A cDNA encoding a lys-plasminogen in which the native plasminogen signalsequence lies adjacent to the Glu(76) residue has been made by deletingthe 75 amino terminal amino acids of glu-plasminogen by loop outmutagenesis using a 35 mer (5'CTGAGAGATACACTTTCTTTTCTCCTTGACCTGAT 3',SEQ ID NO:63). Otherwise, the procedure of Example 3 was generallyfollowed.

Example 31--Construction of Lys-4

In this lys-plasminogen, 77 amino acids between Gly(19) of the signalsequence and Lys(78) of glu-plasminogen were deleted by loop outmutagenesis using a 29 mer (5'CTGAGAGATACACTTTTCCTTGACCTGAT 3', SEQ IDNO:64). Otherwise, the procedure of Example 3 was generally followed.

Example 32--Construction of Lys-5

In this lys-plasminogen, 76 amino acids between Gly(19) of the signalsequence and Lys(77) of glu-plasminogen were deleted by loop outmutagenesis using a 32 mer (5'CTGAGAGATACACTTTCTTTCCTTGACCTGAT 3', SEQID NO:65). Otherwise, the procedure of Example 3 was generally followed.

METHODS

1) Mung Bean Nuclease Digestion

10 units of mung bean nuclease was added to approximately 1 μg DNA whichhad been digested with a restriction enzyme in a buffer containing 30 mMNaOAc pH5.0, 100 mM NaCl, 2 mM ZnCl, 10% glycerol. The mung beannuclease was incubated at 37° for 30 minutes, inactivated for 15 minutesat 67° before being phenol extracted and ethanol precipitated.

2) Oligonucleotide Synthesis

The oligonucleotides were synthesised by automated phosphoramiditechemistry using cyanoethyl phosphoramidites. The methodology is nowwidely used and has been described (Beaucage, S. L. and Caruthers, M. H.Tetrahedron Letters 24, 245 (1981) and Caruthers, M. H. Science 230,281-285 (1985)).

3) Purification of Oligonucleotides

The oligonucleotides were de-protected and removed from the CPG supportby incubation in concentrated NH₃. Typically, 50 mg of CPG carrying 1micromole of oligonucleotide was de-protected by incubation for 5 hoursat 70° in 600 μl of concentrated NH₃. The supernatant was transferred toa fresh tube and the oligomer precipitated with 3 volumes of ethanol.Following centrifugation the pellet was dried and resuspended in 1 ml ofwater. The concentration of crude oligomer was then determined bymeasuring the absorbance at 260 nm.

For gel purification 10 absorbance units of the crude oligonucleotidewas dried down and resuspended in 15 μl of marker dye (90% de-ionisedformamide, 10 mM tris, 10 mM borate, 1 mM EDTA, 0.1% bromophenol blue).The samples were heated at 90° for 1 minute and then loaded onto a 1.2mm thick denaturing polyacrylamide gel with 1.6 mm wide slots. The gelwas prepared from a stock of 15% acrylamide, 0.6% bisacrylamide and 7Murea in 1×TBE and was polymerised with 0.1% ammonium persulphate and0.025% TEMED. The gel was pre-run for 1 hr. The samples were run at 1500V for 4-5 hours. The bands were visualised by UV shadowing and thosecorresponding to the full length product cut out and transferred tomicro-testubes. The oligomers were eluted from the gel slice by soakingin AGEB (0.5M ammonium acetate, 0.01M magnesium acetate and 0.1% SDS)overnight. The AGEB buffer was then transferred to fresh tubes and theoligomer precipitated with three volumes of ethanol at 70° for 15 mins.The precipitate was collected by centrifugion in an Eppendorf microfugefor 10 mins, the pellet washed in 80% ethanol, the purified oligomerdried, redissolved in 1 ml of water and finally filtered through a 0.45micron micro-filter. (The word EPPENDORF is a trade mark.) Theconcentration of purified product was measured by determining itsabsorbance at 260 nm.

4) Kinasing of Oligomers

100 pmole of oligomer was dried down and resuspended in 20 μl kinasebuffer (70 mM Tris pH 7.6, 10 mM MgCl₂, 1 mM ATP, 0.2 mM spermidine, 0.5mM dithiothreitol). 10 u of T4 polynucleotide kinase was added and themixture incubated at 37° for 30 mins. The kinase was then inactivated byheating at 70° for 10 mins.

5) Dideoxy Sequencing

The protocol used was essentially as has been described (Biggin, M. D.,Gibson, T. J., Hong, G. F. P.N.A.S. 80 3963-3965 (1983). Whereappropriate the method was modified to allow sequencing on plasmid DNAas has been described (Guo, L-H., Wu R Nucleic Acids Research 115521-5540 (1983).

6) Transformation

Transformation was accomplished using standard procedures. The strainused as a recipient in the cloning using plasmid vectors was HW87 whichhas the following genotype:

araD139(ara-leu)de17697 (lacIPOZY)de174 galU galK hsdR rpsL srl recA56

RZ1032 is a derivative of E. coli that lacks two enzymes of DNAmetabolism: (a) dUTPase (dut) which results in a high concentration ofintracellular dUTP, and (b) uracil N-glycosylase (ung) which isresponsible for removing mis incorporated uracils from DNA (Kunkel etal, Methods in Enzymol., 154, 367-382 (1987)). Its principal benefit isthat these mutations lead to a higher frequency of mutants in sitedirected mutagenesis. RZ1032 has the following genotype:

HfrKL16PO/45[lysA961-62), dutl, ungl, thil, re[A], Zbd-279::Tn10, supE44

JM103 is a standard recipient strain for manipulations involving M13based vectors.

7) Site Directed Mutagenesis

Kinased mutagenesis primer (2.5 pmole) was annealed to the singlestranded template DNA, which was prepared using RZ1032 as host, (1 μg)in a final reaction mix of 10 μl containing 70 mM Tris, 10 mM MgCl₂. Thereaction mixture in a polypropylene micro-testube (EPPENDORF) was placedin a beaker containing 250 ml of water at 70° C. for 3 minutes followedby 37° C. for 30 minutes. The annealed mixture was then placed on iceand the following reagents added: 1 μl of 10×TM (700 mM Tris, 100 mMMgCl₂ pH7.6), 1 μl of a mixture of all 4 deoxyribonucleotidetriphosphates each at 5 mM, 2 μl of T4 DNA ligase (100 u), 0.5 μl Klenowfragment of DNA polymerase and 4.5 μl of water. The polymerase reactionmixture was then incubated at 15° for 4-16 hrs. After the reaction wascomplete, 180 μl of TE (10 mM Tris, 1 mM EDTA pH8.0) was added and themutagenesis mixture stored at -20° C.

For the isolation of mutant clones the mixture was then transformed intothe recipient JM103 as follows. A 5 ml overnight culture of JM103 in2×YT (1.6% Bactotryptone, 1% Yeast Extract, 1% NaCl) was diluted 1 in a100 into 50 ml of pre-warmed 2×YT. The culture was grown at 37° withaeration until the A₆₀₀ reached 0.4. The cells were pelleted andresuspended in 0.5 vol of 50 mM CaCl₂ and kept on ice for 15 mins. Thecells were then re-pelleted at 4° and resuspended in 2.5 ml cold 50 mMCaCl₂. For the transfection, 0.25, 1, 2, 5, 20 and 50 μl aliquots of themutagenesis mixture were added to 200 μl of competent cells which werekept on ice for 30 mins. The cells were then heated shocked at 42° for 2mins. To each tube was then added 3.5 ml of YT soft agar containing 0.2ml of a late exponential culture of JM103, the contents were mixedbriefly and then poured onto the surface of a pre-warmed platecontaining 2×YT solidified with 1.5% agar. The soft agar layer wasallowed to set and the plates then incubated at 37° overnight.

Single stranded DNA was then prepared from isolated clone as follows:Single plaques were picked into 4 ml of 2×YT that had been seeded with10 μl of a fresh overnight culture of JM103 in 2×YT. The culture wasshaken vigorously for 6 hrs. 0.5 ml of the culture was then removed andadded to 0.5 ml of 50% glycerol to give a reference stock that wasstored at -20°. The remaining culture was centrifuged to remove thecells and 1 ml of supernatant carrying the phage particles wastransferred to a fresh EPPENDORF tube. 250 μl of 20% PEG6000, 250 mMNaCl was then added, mixed and the tubes incubated on ice for 15 mins.The phage were then pelleted at 10,000 rpm for 10 mins, the supernatantdiscarded and the tubes re-centrifuged to collect the final traces ofPEG solution which could then be removed and discarded. The phage pelletwas thoroughly resuspended in 200 μl of TEN (10 mM Tris, 1 mM EDTA, 0.3MNaOAc). The DNA was isolated by extraction with an equal volume of Trissaturated phenol. The phases were separated by a brief centrifugationand the aqueous phase transferred to a clean tube. The DNA wasre-extracted with a mixture of 100 μl of phenol, 100 μl chloroform andthe phases again separated by centrifugation. Traces of phenol wereremoved by three subsequent extractions with chloroform and the DNAfinally isolated by precipitation with 2.5 volumes of ethanol at -20°overnight. The DNA was pelleted at 10,000 rpm for 10 min, washed in 70%ethanol, dried and finally resuspended in 50 μl of TE.

8) Electroporation

Chinese hamster ovary cells (CHO) or the mouse myeloma cell linep3×63-Ag8.653 were grown and harvested in mid log growth phase. Thecells were washed and resuspended in PBS and a viable cell count wasmade. The cells were then pelleted and resuspended at 1×10⁷ cells/ml. 40μg of linearised DNA was added to 1 ml of cells and allowed to stand onice for 15 mins. One pulse of 800 V/25 μF was administered to the cellsusing a commercially available electroporation apparatus (BIORAD GENEPULSER--trade mark). The cells were incubated on ice for a further 15mins and then plated into either 10×96 well plates with 200 μl ofconditioned medium per well (DMEM, 5% FCS, Pen/Strep, glutamine) or10×15 cm dishes with 15 mls medium in each dish and incubated overnight.After 24 hrs the medium was removed and replaced with selective mediacontaining xanthine (250 μg/ml), mycophenolic acid (5 μg/ml) and1×hypoxanthine-thymidine (HT). The cells were fed every third day.

After about 14 days gpt resistant colonies are evident in some of thewells and on the plates. The plates were screened for plasminogen byremoving an aliquot of medium from each well or plate and assayed usingan ELISA assay. Clones producing plasminogen were scaled up and theexpression level monitored to allow the selection of the best producer.

9) ELISA for Human Plasminogen

ELISA plates (Pro-Bind, Falcon) are coated with 50 μl/well of goatanti-human plasminogen serum (Sigma) diluted 1:1000 in coating buffer(4.0 g Na₂ CO₃ (10.H₂ O), 2.93 g NaHCO₃ per liter H₂ O, pH 9.6) andincubated overnight at 4° C. Coating solution is then removed and platesare blocked by incubating with 50 μl/well of PBS/0.1% casein at roomtemperature for 15 minutes. Plates are then washed 3 times withPBS/0.05% Tween 20. Samples of plasminogen or standards diluted inPBS/Tween are added to the plate and incubated at room temperature for 2hours. The plates are then washed 3 times with PBS/Tween and then 50μl/well of a 1:1000 dilution in PBS/Tween of a monoclonal antihumanplasminogen antibody (American Diagnostica, New York, USA) is added andincubated at room temperature for 1 hour. The plates are again washed 3times with PBS/Tween and then 50 μl/well of horse radish peroxidaseconjugated goat anti-mouse IgG (Sigma) is added and incubated at roomtemperature for 1 hour. The plates are washed 5 times with PBS/Tween andthen incubated with 100 μl/well of peroxidase substrate (0.1M sodiumacetate/citric acid buffer pH 6.0 containing 100 mg/liter3,3',5,5'-tetramethyl benzidine and 13 mM H₂ O₂. The reaction is stoppedafter approximately 5 minutes by the addition of 25 μl/well of 2.5Msulphuric acid and the absorbance at 450 nm read on a platereader.

10) Purification of Plasminogen Variants

Plasminogen variants are purified in a single step by chromatography onlysine SEPHAROSE 4B (Pharmacia). A column is equilibrated with at least10 column volumes of 0.05M sodium phosphate buffer pH 7.5. The column isloaded with conditioned medium at a ratio of 1 ml resin per 0.6 mg ofplasminogen variant as determined by ELISA using human glu-plasminogenas standard. Typically 400 ml of conditioned medium containingplasminogen are applied to a 10 ml column (H:D=4) at a linear flow rateof 56 ml/cm/h at 4° C. After loading is complete, the column is washedwith a minimum of 5 column volumes of 0.05M phosphate buffer pH 7.5containing 0.5M NaCl until non-specifically bound protein ceases to beeluted. Desorption of bound plasminogen is achieved by the applicationof 0.2M epsilon-amino-caproic acid in de-ionised water pH 7.0. Elutionrequires 2 column volumes and is carried out at a linear flow rate of 17ml/cm/h. Following analysis by SDS PAGE to check purity,epsilon-amino-caproic acid is subsequently removed and replaced with asuitable buffer, eg Tris, PBS, HEPES or acetate, by chromatography onpre-packed, disposable, PD10 columns containing SEPHADEX G-25M(Pharmacia). (The word SEPHADEX is a trade mark.) Typically, 2.5 ml ofeach plasminogen mutant at a concentration of 0.3 mg/ml are processed inaccordance with the manufacturers' instructions. Fractions containingplasminogen, as determined by A₂₈₀ are then pooled.

11) Cleavage

Plasminogen analogues are assessed for susceptibility to cleavage byproteolytic activators using SDS PAGE under reducing conditions. Typicalincubation volumes of 0.125 ml in 100 mM Tris HCl pH 7.4 and 1 mM Ca²⁺consist of plasminogen analogue, at concentrations shown in theexamples, and the activators Factor Xa or thrombin, at concentrationsshown in the examples. Incubations are performed at 37° C. Controlincubations are performed under the same conditions in the absence ofactivators. The activation reactions were stopped by precipitating theprotein by the addition of trichloroacetic acid to a final concentrationof 20% and standing at 4° C. for >4 hours. The precipitates were thenpelleted, washed with acetone and resuspended in SDS PAGE sample buffer(0.1 m Tris pH6.8, 10% glycerol, 1% SDS, 0.5% mercaptoethanol and 0.05%bromophenol blue). The samples were analysed either on 8-25% gradientgels or 12% gels. The resulting gels were analysed using a SHIMADZU GelScanner which scans the gel and calculates the concentration of proteinin bands by determining the area under the peaks. (The word SHIMADZU isatrade mark.) The rate of cleavage of plasminogen was thus determined bymeasuring the disappearance of the plasminogen band at approximately 92kDa and the appearance of the plasmin heavy chain band at approximately66 kDa.

12) Activation

12.1 Fibrin Clot Lysis Assay

In the fibrin lysis assay, plasmin activity is detected by theappearance of a zone of clearance developing (due to fibrin dissolution)in a fibrin-agarose gel. The gel is made in a 1% low gelling temperatureagarose gel, buffered in 0.1M Tris HCl pH7.4, 0.15M NaCl, 2 mM CaCl₂ byadding plasminogen-free fibrinogen dissolved in 0.9%(w/v) NaCl, to afinal concentration of 1 mg/ml. 6 units of thrombin are added to convertthe fibrinogen to fibrin and the solution is then poured onto a sheet ofGEL-BOND and left to set. (The expression GEL-BOND is atrade mark.)Before use, wells are punched in the gel and the agarose plugs areremoved. Samples of 5-10 μl are loaded into the wells and the gel isincubated in a humidity chamber at 37° C. overnight (17-20 hours), orfor an appropriate time for a zone of lysis to appear. The gel is thensoaked in 7.5% acetic acid for 1 hour, stained in fast green (2%solution) for 1-10 minutes and then destained with 40% methanol, 10%acetic acid for at least 2 hours. The gel is then drained and placed at37° C. overnight to dry. The diameter of the zones of lysis can bemeasured and compared to those made by the standards e.g. wild typeplasminogen activated with tPA or u-PA.

12.2 Direct Chromogenic Assay and Time Course of Activation

Plasminogen analogue (12.5 μg) was incubated with thrombin (2.8 μg) at37° C. in 125 μl of a buffer containing 100 mM Tris HCl pH 7.4 and 1 mMCaCl₂. Aliquots were removed at intervals and assayed for plasmincontent in a chromogenic assay as described below. When thrombin wasused as activator the thrombin inhibitor hirudin was added in slightmolar excess to stop the activation reaction and the samples were storedat -70° C. When Factor Xa was used as activator samples were immediatelysnap frozen to stop the activation reaction. Plasmin was measured usingcleavage of the tripeptide chromogenic substrate, S2251 (Kabi). Aliquotsof the sample (25 μl ) were mixed with 75 μl buffer (50 mM Tris HCl,0.1M EDTA, 0.0005% Triton X100, pH 8.0) containing 0.6 mM S2251, in 96well plates (Costar). The plates were incubated at 37° C. for 2 hours.The reaction was terminated by adding 25 μl 0.5M acetic acid and theabsorbance read at 405 nm using an automatic plate reader (Dynatech).Quantitation was performed by comparison with a standard plasminpreparation.

12.3 Linked Chromogenic Assay

A modification of the chromogenic assay was developed to measure thetime course of activation of mutant plasminogens more directly. In thisassay, mutant plasminogen and activator are incubated together in thepresence of S2251 and plasmin produced by activation directly cleavesthe chromogenic substrate leading to an increase in absorbance at 405nm. The assay was performed in a total volume of 880 μl in a buffercontaining 50 mM Tris HCl, 0.1 mM EDTA, 0.005% Triton X100 and 0.1% HSA.The chromogenic substrate S2251 was added to a final concentration of0.35 mg/ml and the mutant protein concentration used was 3 μg/ml. In thecase of thrombin activation, thrombin was added to a final concentrationof 1 or 0.2 μg/ml. Factor Xa was added to a final concentration of 1.5or 0.3 μg/ml. Aliquots of 100 μl of the reaction were removed atintervals and added to 25 μl 4% acetic acid, in microtitre plates, tostop the reaction. At the completion of the time course the plates areread on a microplate reader at a wavelength of 405 nm. No attempt wasmade to quantify plasmin generation in this assay.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 67                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2753 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..2753                                                         (D) OTHER INFORMATION: /note="FIG. 2 Plasminogen cDNA                         sequence"                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: sig.sub.-- peptide                                              (B) LOCATION: 65..121                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 122..2494                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 65..2494                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 54..55                                                          (D) OTHER INFORMATION: /note="BalI site"                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 2564..2565                                                      (D) OTHER INFORMATION: /note="SphI site"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GATGTAAGTCAACAACATCCTGGGATTGGGACCCACTTTCTGGGCACTGCTGGCCAGTCC60                CAAAATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCTGAAA109                          MetGluHisLysGluValValLeuLeuLeuLeuLeuPheLeuLys                                 19-15-10-5                                                                    TCAGGTCAAGGAGAGCCTCTGGATGACTATGTGAATACCCAGGGGGCT157                           SerGlyGlnGlyGluProLeuAspAspTyrValAsnThrGlnGlyAla                              1510                                                                          TCACTGTTCAGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAA205                           SerLeuPheSerValThrLysLysGlnLeuGlyAlaGlySerIleGlu                              152025                                                                        GAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTCACCTGCAGGGCA253                           GluCysAlaAlaLysCysGluGluAspGluGluPheThrCysArgAla                              303540                                                                        TTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAAC301                           PheGlnTyrHisSerLysGluGlnGlnCysValIleMetAlaGluAsn                              45505560                                                                      AGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAA349                           ArgLysSerSerIleIleIleArgMetArgAspValValLeuPheGlu                              657075                                                                        AAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTAC397                           LysLysValTyrLeuSerGluCysLysThrGlyAsnGlyLysAsnTyr                              808590                                                                        AGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGG445                           ArgGlyThrMetSerLysThrLysAsnGlyIleThrCysGlnLysTrp                              95100105                                                                      AGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCC493                           SerSerThrSerProHisArgProArgPheSerProAlaThrHisPro                              110115120                                                                     TCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCG541                           SerGluGlyLeuGluGluAsnTyrCysArgAsnProAspAsnAspPro                              125130135140                                                                  CAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTAC589                           GlnGlyProTrpCysTyrThrThrAspProGluLysArgTyrAspTyr                              145150155                                                                     TGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAA637                           CysAspIleLeuGluCysGluGluGluCysMetHisCysSerGlyGlu                              160165170                                                                     AACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAG685                           AsnTyrAspGlyLysIleSerLysThrMetSerGlyLeuGluCysGln                              175180185                                                                     GCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAA733                           AlaTrpAspSerGlnSerProHisAlaHisGlyTyrIleProSerLys                              190195200                                                                     TTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGG781                           PheProAsnLysAsnLeuLysLysAsnTyrCysArgAsnProAspArg                              205210215220                                                                  GAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAA829                           GluLeuArgProTrpCysPheThrThrAspProAsnLysArgTrpGlu                              225230235                                                                     CTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCC877                           LeuCysAspIleProArgCysThrThrProProProSerSerGlyPro                              240245250                                                                     ACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTG925                           ThrTyrGlnCysLeuLysGlyThrGlyGluAsnTyrArgGlyAsnVal                              255260265                                                                     GCTGTTACCGTGTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACC973                           AlaValThrValSerGlyHisThrCysGlnHisTrpSerAlaGlnThr                              270275280                                                                     CCTCACACACATAACAGGACACCAGAAAACTTTCCCTGCAAAAATTTG1021                          ProHisThrHisAsnArgThrProGluAsnPheProCysLysAsnLeu                              285290295300                                                                  GATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGC1069                          AspGluAsnTyrCysArgAsnProAspGlyLysArgAlaProTrpCys                              305310315                                                                     CATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCC1117                          HisThrThrAsnSerGlnValArgTrpGluTyrCysLysIleProSer                              320325330                                                                     TGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCA1165                          CysAspSerSerProValSerThrGluGlnLeuAlaProThrAlaPro                              335340345                                                                     CCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAG1213                          ProGluLeuThrProValValGlnAspCysTyrHisGlyAspGlyGln                              350355360                                                                     AGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAG1261                          SerTyrArgGlyThrSerSerThrThrThrThrGlyLysLysCysGln                              365370375380                                                                  TCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAAC1309                          SerTrpSerSerMetThrProHisArgHisGlnLysThrProGluAsn                              385390395                                                                     TACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCC1357                          TyrProAsnAlaGlyLeuThrMetAsnTyrCysArgAsnProAspAla                              400405410                                                                     GATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAG1405                          AspLysGlyProTrpCysPheThrThrAspProSerValArgTrpGlu                              415420425                                                                     TACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCA1453                          TyrCysAsnLeuLysLysCysSerGlyThrGluAlaSerValValAla                              430435440                                                                     CCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAA1501                          ProProProValValLeuLeuProAspValGluThrProSerGluGlu                              445450455460                                                                  GACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACC1549                          AspCysMetPheGlyAsnGlyLysGlyTyrArgGlyLysArgAlaThr                              465470475                                                                     ACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCAT1597                          ThrValThrGlyThrProCysGlnAspTrpAlaAlaGlnGluProHis                              480485490                                                                     AGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAA1645                          ArgHisSerIlePheThrProGluThrAsnProArgAlaGlyLeuGlu                              495500505                                                                     AAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGC1693                          LysAsnTyrCysArgAsnProAspGlyAspValGlyGlyProTrpCys                              510515520                                                                     TACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAG1741                          TyrThrThrAsnProArgLysLeuTyrAspTyrCysAspValProGln                              525530535540                                                                  TGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAG1789                          CysAlaAlaProSerPheAspCysGlyLysProGlnValGluProLys                              545550555                                                                     AAATGTCCTGGAAGGGTTGTAGGGGGGTGTGTGGCCCACCCACATTCC1837                          LysCysProGlyArgValValGlyGlyCysValAlaHisProHisSer                              560565570                                                                     TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGT1885                          TrpProTrpGlnValSerLeuArgThrArgPheGlyMetHisPheCys                              575580585                                                                     GGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGC1933                          GlyGlyThrLeuIleSerProGluTrpValLeuThrAlaAlaHisCys                              590595600                                                                     TTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCA1981                          LeuGluLysSerProArgProSerSerTyrLysValIleLeuGlyAla                              605610615620                                                                  CACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCT2029                          HisGlnGluValAsnLeuGluProHisValGlnGluIleGluValSer                              625630635                                                                     AGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA2077                          ArgLeuPheLeuGluProThrArgLysAspIleAlaLeuLeuLysLeu                              640645650                                                                     AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCA2125                          SerSerProAlaValIleThrAspLysValIleProAlaCysLeuPro                              655660665                                                                     TCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGC2173                          SerProAsnTyrValValAlaAspArgThrGluCysPheIleThrGly                              670675680                                                                     TGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCC2221                          TrpGlyGluThrGlnGlyThrPheGlyAlaGlyLeuLeuLysGluAla                              685690695700                                                                  CAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTG2269                          GlnLeuProValIleGluAsnLysValCysAsnArgTyrGluPheLeu                              705710715                                                                     AATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA2317                          AsnGlyArgValGlnSerThrGluLeuCysAlaGlyHisLeuAlaGly                              720725730                                                                     GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTC2365                          GlyThrAspSerCysGlnGlyAspSerGlyGlyProLeuValCysPhe                              735740745                                                                     GAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGC2413                          GluLysAspLysTyrIleLeuGlnGlyValThrSerTrpGlyLeuGly                              750755760                                                                     TGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTT2461                          CysAlaArgProAsnLysProGlyValTyrValArgValSerArgPhe                              765770775780                                                                  GTTACTTGGATTGAGGGAGTGATGAGAAATAATTAATTGGACGGGAGACAGAG2514                     ValThrTrpIleGluGlyValMetArgAsnAsn                                             785790                                                                        TGACGCACTGACTCACCTAGAGGCTGGAACGTGGGTAGGGATTTAGCATGCTGGAAATAA2574              CTGGCAGTAATCAAACGAAGACACTGTCCCCAGCTACCAGCTACGCCAAACCTCGGCATT2634              TTTTGTGTTATTTTCTGACTGCTGGATTCTGTAGTAAGGTGACATAGCTATGACATTTGT2694              TAAAAATAAACTCTGTACTTAACTTTGATTTGAGTAAATTTTGGTTTTGGTCTTCAACA2753               (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 810 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGluHisLysGluValValLeuLeuLeuLeuLeuPheLeuLysSer                              19- 15-10-5                                                                   GlyGlnGlyGluProLeuAspAspTyrValAsnThrGlnGlyAlaSer                              1510                                                                          LeuPheSerValThrLysLysGlnLeuGlyAlaGlySerIleGluGlu                              152025                                                                        CysAlaAlaLysCysGluGluAspGluGluPheThrCysArgAlaPhe                              30354045                                                                      GlnTyrHisSerLysGluGlnGlnCysValIleMetAlaGluAsnArg                              505560                                                                        LysSerSerIleIleIleArgMetArgAspValValLeuPheGluLys                              657075                                                                        LysValTyrLeuSerGluCysLysThrGlyAsnGlyLysAsnTyrArg                              808590                                                                        GlyThrMetSerLysThrLysAsnGlyIleThrCysGlnLysTrpSer                              95100105                                                                      SerThrSerProHisArgProArgPheSerProAlaThrHisProSer                              110115120125                                                                  GluGlyLeuGluGluAsnTyrCysArgAsnProAspAsnAspProGln                              130135140                                                                     GlyProTrpCysTyrThrThrAspProGluLysArgTyrAspTyrCys                              145150155                                                                     AspIleLeuGluCysGluGluGluCysMetHisCysSerGlyGluAsn                              160165170                                                                     TyrAspGlyLysIleSerLysThrMetSerGlyLeuGluCysGlnAla                              175180185                                                                     TrpAspSerGlnSerProHisAlaHisGlyTyrIleProSerLysPhe                              190195200205                                                                  ProAsnLysAsnLeuLysLysAsnTyrCysArgAsnProAspArgGlu                              210215220                                                                     LeuArgProTrpCysPheThrThrAspProAsnLysArgTrpGluLeu                              225230235                                                                     CysAspIleProArgCysThrThrProProProSerSerGlyProThr                              240245250                                                                     TyrGlnCysLeuLysGlyThrGlyGluAsnTyrArgGlyAsnValAla                              255260265                                                                     ValThrValSerGlyHisThrCysGlnHisTrpSerAlaGlnThrPro                              270275280285                                                                  HisThrHisAsnArgThrProGluAsnPheProCysLysAsnLeuAsp                              290295300                                                                     GluAsnTyrCysArgAsnProAspGlyLysArgAlaProTrpCysHis                              305310315                                                                     ThrThrAsnSerGlnValArgTrpGluTyrCysLysIleProSerCys                              320325330                                                                     AspSerSerProValSerThrGluGlnLeuAlaProThrAlaProPro                              335340345                                                                     GluLeuThrProValValGlnAspCysTyrHisGlyAspGlyGlnSer                              350355360365                                                                  TyrArgGlyThrSerSerThrThrThrThrGlyLysLysCysGlnSer                              370375380                                                                     TrpSerSerMetThrProHisArgHisGlnLysThrProGluAsnTyr                              385390395                                                                     ProAsnAlaGlyLeuThrMetAsnTyrCysArgAsnProAspAlaAsp                              400405410                                                                     LysGlyProTrpCysPheThrThrAspProSerValArgTrpGluTyr                              415420425                                                                     CysAsnLeuLysLysCysSerGlyThrGluAlaSerValValAlaPro                              430435440445                                                                  ProProValValLeuLeuProAspValGluThrProSerGluGluAsp                              450455460                                                                     CysMetPheGlyAsnGlyLysGlyTyrArgGlyLysArgAlaThrThr                              465470475                                                                     ValThrGlyThrProCysGlnAspTrpAlaAlaGlnGluProHisArg                              480485490                                                                     HisSerIlePheThrProGluThrAsnProArgAlaGlyLeuGluLys                              495500505                                                                     AsnTyrCysArgAsnProAspGlyAspValGlyGlyProTrpCysTyr                              510515520525                                                                  ThrThrAsnProArgLysLeuTyrAspTyrCysAspValProGlnCys                              530535540                                                                     AlaAlaProSerPheAspCysGlyLysProGlnValGluProLysLys                              545550555                                                                     CysProGlyArgValValGlyGlyCysValAlaHisProHisSerTrp                              560565570                                                                     ProTrpGlnValSerLeuArgThrArgPheGlyMetHisPheCysGly                              575580585                                                                     GlyThrLeuIleSerProGluTrpValLeuThrAlaAlaHisCysLeu                              590595600605                                                                  GluLysSerProArgProSerSerTyrLysValIleLeuGlyAlaHis                              610615620                                                                     GlnGluValAsnLeuGluProHisValGlnGluIleGluValSerArg                              625630635                                                                     LeuPheLeuGluProThrArgLysAspIleAlaLeuLeuLysLeuSer                              640645650                                                                     SerProAlaValIleThrAspLysValIleProAlaCysLeuProSer                              655660665                                                                     ProAsnTyrValValAlaAspArgThrGluCysPheIleThrGlyTrp                              670675680685                                                                  GlyGluThrGlnGlyThrPheGlyAlaGlyLeuLeuLysGluAlaGln                              690695700                                                                     LeuProValIleGluAsnLysValCysAsnArgTyrGluPheLeuAsn                              705710715                                                                     GlyArgValGlnSerThrGluLeuCysAlaGlyHisLeuAlaGlyGly                              720725730                                                                     ThrAspSerCysGlnGlyAspSerGlyGlyProLeuValCysPheGlu                              735740745                                                                     LysAspLysTyrIleLeuGlnGlyValThrSerTrpGlyLeuGlyCys                              750755760765                                                                  AlaArgProAsnLysProGlyValTyrValArgValSerArgPheVal                              770775780                                                                     ThrTrpIleGluGlyValMetArgAsnAsn                                                785790                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..36                                                           (D) OTHER INFORMATION: /note="Fig 4 WT cleavage site                          sequence, Factor Xa series, residues are 555-566"                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..36                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..36                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGT36                                        ProLysLysCysProGlyArgValValGlyGlyCys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ProLysLysCysProGlyArgValValGlyGlyCys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..39                                                           (D) OTHER INFORMATION: /note="Fig 4 X1 analog"                                (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..39                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCGAAGAAATGTATCGAGGGAAGGGTTGTGGGGGGGTGT39                                     ProLysLysCysIleGluGlyArgValValGlyGlyCys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ProLysLysCysIleGluGlyArgValValGlyGlyCys                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..42                                                           (D) OTHER INFORMATION: /note="FIG. 4 X2 analog"                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..42                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..42                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CCGAAGAAATGTGGCATCGAGGGAAGGGTTGTGGGGGGGTGT42                                  ProLysLysCysGlyIleGluGlyArgValValGlyGlyCys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ProLysLysCysGlyIleGluGlyArgValValGlyGlyCys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..45                                                           (D) OTHER INFORMATION: /note="FIG. 4 X3 analog"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CCGAAGAAATGTGGTGCAATAGAGGGAAGGGTTGTGGGGGGGTGT45                               ProLysLysCysGlyAlaIleGluGlyArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ProLysLysCysGlyAlaIleGluGlyArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..45                                                           (D) OTHER INFORMATION: /note="FIG. 4 X5 analog"                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..45                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..45                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CCGAAGAAATGTGGTTACATAGACGGAAGGGTTGTGGGGGGGTGT45                               ProLysLysCysGlyTyrIleAspGlyArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ProLysLysCysGlyTyrIleAspGlyArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..45                                                           (D) OTHER INFORMATION: /note="FIG. 4 X6 analog"                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..45                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..45                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CCGAAGAAATGTGGTTACATAGACGGAAGGATTGTGGGGGGGTGT45                               ProLysLysCysGlyTyrIleAspGlyArgIleValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ProLysLysCysGlyTyrIleAspGlyArgIleValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..42                                                           (D) OTHER INFORMATION: /note="FIG. 5 WT Cleavage site -                       Thrombin series, segment corresponds to residue                               553-566"                                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..42                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..42                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGT42                                  ValGluProLysLysCysProGlyArgValValGlyGlyCys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ValGluProLysLysCysProGlyArgValValGlyGlyCys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..42                                                           (D) OTHER INFORMATION: /note="FIG. 5 T1 analog"                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..42                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..42                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GTGGAGCCGAAGAAATGTGGTCCTAGGGTTGTGGGGGGGTGT42                                  ValGluProLysLysCysGlyProArgValValGlyGlyCys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ValGluProLysLysCysGlyProArgValValGlyGlyCys                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..45                                                           (D) OTHER INFORMATION: /note="FIG. 4 T2 analog"                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..45                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..45                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GTGGAGCCGAAGAAATGTGGTGGTCCAAGGGTTGTGGGGGGGTGT45                               ValGluProLysLysCysGlyGlyProArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ValGluProLysLysCysGlyGlyProArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..48                                                           (D) OTHER INFORMATION: /note="FIG. 5 T6 analog"                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..48                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..48                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CTGGAGCCGGAGCTATGTGGAGTTGTGCCTAGGGGAGTGGGGGGGTGT48                            LeuGluProGluLeuCysGlyValValProArgGlyValGlyGlyCys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      LeuGluProGluLeuCysGlyValValProArgGlyValGlyGlyCys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..48                                                           (D) OTHER INFORMATION: /note="FIG. 5 T7 analog"                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..48                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..48                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CTGGAGCCGCAACTATGTGGAGTTGTGCCTAGGGGAGTGGGGGGGTGT48                            LeuGluProGlnLeuCysGlyValValProArgGlyValGlyGlyCys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      LeuGluProGlnLeuCysGlyValValProArgGlyValGlyGlyCys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..60                                                           (D) OTHER INFORMATION: /note="FIG. 5 T8 analog"                               (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..60                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..60                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GTGGAGCCGAAGAAATGTGTAGAACTACAAGGAGTAGTGCCTAGGGGA48                            ValGluProLysLysCysValGluLeuGlnGlyValValProArgGly                              151015                                                                        GTGGGGGGGTGT60                                                                ValGlyGlyCys                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      ValGluProLysLysCysValGluLeuGlnGlyValValProArgGly                              151015                                                                        ValGlyGlyCys                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..45                                                           (D) OTHER INFORMATION: /note="FIG. 5 T13 analog"                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..45                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..45                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GTGGAGCCGAAGAAATGTGTTGTACCTAGGGTTGTGGGGGGGTGT45                               ValGluProLysLysCysValValProArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      ValGluProLysLysCysValValProArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..45                                                           (D) OTHER INFORMATION: /note="FIG. 5 T14 analog"                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..45                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..45                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GTGGAGCCGAAGAAATGTGGATACCCTAGGGTTGTGGGGGGGTGT45                               ValGluProLysLysCysGlyTyrProArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      ValGluProLysLysCysGlyTyrProArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..45                                                           (D) OTHER INFORMATION: /note="FIG. 5 T17 analog"                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..45                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..45                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GTGGAGCCGAAGAAATGTCCTAGTGGAAGGGTTGTGGGGGGGTGT45                               ValGluProLysLysCysProSerGlyArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      ValGluProLysLysCysProSerGlyArgValValGlyGlyCys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..60                                                           (D) OTHER INFORMATION: /note="FIG. 5 T19 analog"                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..60                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..60                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GTGGAGCCGAAGAAATGTGTAGAATTGCAGGGAGTAGTCCCAAGGGTT48                            ValGluProLysLysCysValGluLeuGlnGlyValValProArgVal                              151015                                                                        GTGGGGGGGTGT60                                                                ValGlyGlyCys                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      ValGluProLysLysCysValGluLeuGlnGlyValValProArgVal                              151015                                                                        ValGlyGlyCys                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..54                                                           (D) OTHER INFORMATION: /note="FIG. 5 T20 analog"                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..54                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..54                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      GTGGAGCCGAAGAAATGTGTAGAATTGCAGGGAGTAGTCCCAAGGGGG48                            ValGluProLysLysCysValGluLeuGlnGlyValValProArgGly                              151015                                                                        GGGTGT54                                                                      GlyCys                                                                        (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      ValGluProLysLysCysValGluLeuGlnGlyValValProArgGly                              151015                                                                        GlyCys                                                                        (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..48                                                           (D) OTHER INFORMATION: /note="FIG. 5 T21 analog"                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..48                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..48                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      CTGGAGCCGGAGCTATGTGGAGTTGTGCCTAGGGTAGTGGGGGGGTGT48                            LeuGluProGluLeuCysGlyValValProArgValValGlyGlyCys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      LeuGluProGluLeuCysGlyValValProArgValValGlyGlyCys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..48                                                           (D) OTHER INFORMATION: /note="FIG. 5 T22 analog"                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..48                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 1..48                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      CTGGAGCCGCAACTATGTGGAGTTGTGCCTAGGGTAGTGGGGGGGTGT48                            LeuGluProGlnLeuCysGlyValValProArgValValGlyGlyCys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      LeuGluProGlnLeuCysGlyValValProArgValValGlyGlyCys                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 105 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AGCTTCCACCATGAAGTGCTCCTGGGTGATCTTCTTCCTGATGGCCGTGGTGACCGGCGT60                GAACTCGCGAGATCTAGAGTCGACCTGCAGGATATCGAATTCATT105                              (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 105 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GATCAATGAATTCGATATCCTGCAGGTCGACTCTAGATCTCGCGAGTTCACGCCGGTCAC60                CACGGCCATCAGGAAGAAGATCACCCAGGAGCACTTCATGGTGGA105                              (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      TATGAAGACGTCGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGC60                CTCTGCATAAATAAAAAAAATTAGTCAGGG90                                              (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      CGCCCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTAT60                TCCAGAAGTAGTGAGGAGGCGACGTCTTCA90                                              (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      AAGCGGCCGCGGCCATGCCGGCCACTAGTCTCGAGTT37                                       (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      AACTCGAGACTAGTGGCCGGCATGGCCGCGGCCGCTT37                                       (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      CCCTTCCCTCGATACATTTCT21                                                       (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      CCTTCCCTCGATGCCACATTTC22                                                      (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      CCCCCCCACAACCCTTCCCTCTATTGCACCACATTTCTTCGGCTCCAC48                            (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      CCCCCCCACAACCCTTCCGTCTATGTAACCACATTTCTTCGGCTCCAC48                            (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      CACACCCCCCCACAATCCTTCCGTCTATGTAACCACATTTCTTCGGCTCCAC52                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      CAACCCTTGGACCACATTTCT21                                                       (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      ACCCTTGGACCACCACATTTCT22                                                      (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GGGCCACACACCCCCCCACTCCCCTAGGCACAACTCCACATAGCTCCGGCTCCAGTTGAG60                G61                                                                           (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GGCCACACACCCCCCCACTCCCCTAGGCACAACTCCACATAGTTGCGGCTCCAGTTGAGG60                (2) INFORMATION FOR SEQ ID NO: 56:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:                                     CACACACCCCCCCCTCCCCTAGGCACTACTCCTTGTAGTTCTACACATTTCTTCGGCTCC60                (2) INFORMATION FOR SEQ ID NO: 57:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      CACCCCCCCACAACCCTAGGTACAACACATTTCTTCGGCTC41                                   (2) INFORMATION FOR SEQ ID NO: 58:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:                                     CACCCCCCCACAACCCTAGGGTATCCACATTTCTTCGGCT40                                    (2) INFORMATION FOR SEQ ID NO: 59:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:                                     CACCCCCCCACAACCCTTCCACTAGGACATTTCTTCGG38                                      (2) INFORMATION FOR SEQ ID NO: 60:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:                                     CACACCCCCCCACAACCCTTGGGACTACTCCCTGACATTCTACACATTTCTTCGGCTCCA60                C61                                                                           (2) INFORMATION FOR SEQ ID NO: 61:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:                                     GGCCACACACCCCCCCCTTGGGACTACTCCCTGCAATTCTACACATTTCTTCGGCTCC58                  (2) INFORMATION FOR SEQ ID NO: 62:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:                                     CACCCCCCCACTACCCTAGGCAC23                                                     (2) INFORMATION FOR SEQ ID NO: 63:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:                                     CTGAGAGATACACTTTCTTTTCTCCTTGACCTGAT35                                         (2) INFORMATION FOR SEQ ID NO: 64:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:                                     CTGAGAGATACACTTTTCCTTGACCTGAT29                                               (2) INFORMATION FOR SEQ ID NO: 65:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:                                     CTGAGAGATACACTTTCTTTCCTTGACCTGAT32                                            (2) INFORMATION FOR SEQ ID NO: 66:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:                                     AGCTTCCCGGGATAGGTACCTCG23                                                     (2) INFORMATION FOR SEQ ID NO: 67:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:                                     CGAGGTACCTATCCCGGGA19                                                         __________________________________________________________________________

We claim:
 1. A proteinaceous compound which is activatable by thrombinor Factor Xa enzyme to have fibrinolytic activity; characterized in thatthe compound is a plasminogen analogue, wherein the cleavage sitePro(559) to Val(562) of native plasminogen is altered by substitution ofan amino acid sequence clearable by thrombin or Factor Xa, saidplasminogen analogue being activatable by thrombin or Factor Xa cleavageto have plasmin activity.
 2. A compound as claimed in claim 1, whereinthe enzyme is Factor Xa.
 3. A compound as claimed in claim 2, whereinthe amino acid sequence comprises the sequence P4-P3-Gly-Arg, wherein P4represents a hydrophobic residue and P3 represents an acidic residue. 4.A compound as claimed in claim 3, wherein the hydrophobic residue isisoleucine.
 5. A compound as claimed in claim 1, wherein the enzyme isthrombin.
 6. A compound as claimed in claim 5, wherein the amino acidsequence comprises the sequence P4-P3-Pro-Arg-P1'-P2', wherein each ofP4 and P3 independently represents a hydrophobic residue and each of P1'and P2' independently represents a non-acidic residue.
 7. A compound asclaimed in claim 5, wherein the amino acid sequence comprises thesequence P2-Arg-P1', wherein one of the residues P2 and P1' representsglycine, and the other is any amino acid residue.
 8. A compound asclaimed in claim 5, wherein the amino acid sequence comprises thesequence Gly-Pro-Arg.
 9. A compound as claimed in claim 1 having one ormore amino acid substitutions, additions or deletions between residuesPro(555) and Cys(566) inclusive.
 10. A plasminogen analogue as claimedin claim 1 wherein the cleavage site sequence (SEQ ID NO:6) is: ##STR1##and is in a position corresponding to that of the cleavage site sequencefrom Pro(555) to Cys(566) inclusive of wild-type plasminogen.
 11. Aplasminogen analogue as claimed in claim 1 wherein the cleavage sitesequence (SEQ ID NO:8) is: ##STR2## and is in a position correspondingto that of the cleavage site sequence from Pro(555) to Cys(566)inclusive of wild-type plasminogen.
 12. A plasminogen analogue asclaimed in claim 1 wherein the cleavage site sequence (SEQ ID NO:10) is:##STR3## and is in a position corresponding to that of the cleavage sitesequence from Pro(555) to Cys(566) inclusive of wild-type plasminogen.13. A plasminogen analogue as claimed in claim 1 wherein the cleavagesite sequence (SEQ ID NO:12) is: ##STR4## and is in a positioncorresponding to that of the cleavage site sequence from Pro(555) toCys(566) inclusive of wild-type plasminogen.
 14. A plasminogen analogueas claimed in claim 1 wherein the cleavage site sequence (SEQ ID NO:14)is: ##STR5## and is in a position corresponding to that of the cleavagesite sequence from Pro(555) to Cys (566) inclusive of wild-typeplasminogen.
 15. A plasminogen analogue as claimed in claim 1 whereinthe cleavage site sequence (SEQ ID NO:18) is: ##STR6## and is in aposition corresponding to that of the cleavage site sequence fromVal(553) to Cys (566) inclusive of wild-type plasminogen.
 16. Aplasminogen analogue as claimed in claim 1 wherein the cleavage sitesequence (SEQ ID NO:20) is: ##STR7## and is in a position correspondingto that of the cleavage site sequence from Val(553) to Cys(566)inclusive of wild-type plasminogen.
 17. A plasminogen analogue asclaimed in claim 1 wherein the cleavage site sequence (SEQ ID NO:28) is:##STR8## and is in a position corresponding to that of the cleavage sitesequence from Val(553) to Cys(566) inclusive of wild-type plasminogen.18. A plasminogen analogue as claimed in claim 1 wherein the cleavagesite sequence (SEQ ID NO:32) is: ##STR9## and is in a positioncorresponding to that of the cleavage site sequence from Val(553) toCys(566) inclusive of wild-type plasminogen.
 19. A plasminogen analogueas claimed in claim 1 wherein the cleavage site sequence (SEQ ID NO:34)is: ##STR10## and is in a position corresponding to that of the cleavagesite sequence from Val(553) to Cys(566) inclusive of wild-typeplasminogen.
 20. A proteinaceous compound which is activatable by FactorXa to have fibrinolytic activity; characterized in that the compound isa plasminogen analogue, wherein the cleavage site Pro(559) to Val(562)of native plasminogen is altered by substitution of an amino acidsequence clearable by Factor Xa; said amino acid sequence comprising asequence P4-P3-Gly-Arg, wherein P4 represents a hydrophobic residue andP3 represents an acidic residue; said plasminogen analogue beingactivatable by Factor Xa cleavage to have plasmin activity.
 21. Acompound as claimed claim 20, wherein the hydrophobic residue isisoleucine.
 22. A proteinaceous compound which is activatable bythrombin to have fibrinolytic activity; characterized in that thecompound is a plasminogen analogue, wherein the cleavage site Pro(559)to Val(562) of native plasminogen is altered by substitution of an aminoacid sequence clearable by thrombin; said amino acid sequence comprisinga sequence P4-P3-Pro-Arg-P1'-P2', wherein each of P4 and P3independently represents a hydrophobic residue and each of P1' and P2'independently represents a non-acidic residue; said plasminogen analoguebeing activatable by thrombin cleavage to have plasmin activity.
 23. Aproteinaceous compound which is activatable by thrombin to havefibrinolytic activity; characterized in that the compound is aplasminogen analogue, wherein the cleavage site Pro(559) to Val(562) ofnative plasminogen is altered by substitution of an amino acid sequenceclearable by thrombin; said amino acid sequence comprising a sequenceP2-Arg-P1', wherein one of the residues P2 and P1' represents glycineand the other represents any amino acid residue; said plasminogenanalogue being activatable by thrombin cleavage to have plasminactivity.
 24. A proteinaceous compound which is activatable by thrombinto have fibrinolytic activity; characterized in that the compound is aplasminogen analogue, wherein the cleavage site Pro(559) to Val(562) ofnative plasminogen is altered by substitution of an amino acid sequenceclearable by thrombin; said amino acid sequence comprising a sequenceGly-Pro-Arg; said plasminogen analogue being activatable by thrombincleavage to have plasmin activity.